Connective tissue growth factor antisense oligonucleotides

ABSTRACT

The present invention relates to antisense oligonucleotides that target human CTGF mRNA and inhibit CTGF mRNA expression. Additionally, regions of human CTGF mRNA that are exceptionally sensitive to antisense inhibition are disclosed. Pharmaceutical compositions comprising the antisense oligonucleotides are further disclosed. These compositions are useful for treating disorders and conditions that are associated with or influenced by CTGF expression.

This application claims the benefit of U.S. Provisional Application Ser.No. 61/508,264, filed on 15 Jul. 2011.

FIELD OF THE INVENTION

The present invention provides compounds, compositions and methods formodulating the expression of human connective tissue growth factor(CTGF). In particular, this invention relates to antisenseoligonucleotides (ASOs) capable of modulating human CTGF mRNA expressionthat are useful for treating disorders and conditions involvingdysregulated CTGF expression or that are influenced by the normalexpression of CTGF.

BACKGROUND OF THE INVENTION

CTGF is a 36 kD cysteine-rich, heparin binding, secreted glycoproteinoriginally isolated from the culture media of human umbilical veinendothelial cells. See e.g., Bradham et al. J Cell Biol. (1991) 114;1285-1294; Grotendorst and Bradham, U.S. Pat. No. 5,408,040. CTGFpromotes the proliferation and chemotaxis of various cell types inculture. Additionally, CTGF increases steady-state transcription ofα1(I) collagen, α5 integrin, and fibronectin mRNAs. See e.g., Frazier etal. J Invest Dermatol. (1996) 107:406-411; Shi-wen et al. Exp Cell Res.(2000) 259:213-224; Klagsburn Exp Cell Res. (1977) 105:99-108; Gupta etal. Kidney Int. (2000) 58:1389-1399; Wahab et al. Biochem J. (2001)359(Pt 1):77-87; Uzel et al. J Periodontol. (2001) 72:921-931; and Riserand Cortes Ren Fail. (2001) 23:459-470.

Through the promotion of cellular chemotaxis and proliferation alongwith an increase in expression of extracellular matrix (ECM) components,CTGF plays a role in regulating skeletal development, wound healing, ECMremodeling, fibrosis, tumorigenesis and angiogenesis. For example,elevated CTGF expression has been observed in cirrhotic liver, pulmonaryfibrosis, inflammatory bowel disease, sclerotic skin and keloids,desmoplasia and atherosclerotic plaques. Abraham et al. J Biol Chem.(2000) 275:15220-15225; Dammeier et al. Int. J Biochem Cell Biol. (1998)30:909-922; diMola et al. Ann Surg. (1999) 230(1):63-71; Igarashi et al.J Invest Dermatol. (1996) 106:729-733; Ito et al. Kidney Int. (1998)53:853-861; Williams et al. J. Hepatol. (2000) 32:754-761; Clarkson etal. Curr Opin Nephrol. Hypertens. (1999) 8:543-548; Hinton et al. Eye.(2002): 16:422-428; Gupta et al. Kidney Int. (2000) 58:1389-1399; Riseret al. J Am Soc Nephrol. (2000) 11:25-38.

CTGF is also upregulated in glomerulonephritis, IgA nephropathy, focaland segmental glomerulosclerosis and diabetic nephropathy. See, e.g.,Riser et al. J Am Soc Nephrol. (2000) 11:25-38. An increase in thenumber of cells expressing CTGF mRNA is also observed at sites ofchronic tubulointerstitial damage, with CTGF mRNA levels correlated withthe degree of damage. Ito et al. Kidney Int. (1998) 53:853-861.Additionally, CTGF expression is increased in the glomeruli andtubulointerstium in a variety of renal diseases in association withscarring and sclerosis of renal parenchyma. Elevated levels of CTGF havealso been associated with liver fibrosis, myocardial infarction, andpulmonary fibrosis. For example, in patients with idiopathic pulmonaryfibrosis (IPF), CTGF is highly enriched in biopsies and bronchoalveolarlavage cells. (Ujike et al. Biochem Biophys Res Commun. (2000)277:448-454; Abou-Shady et al. Liver. (2000) 20:296-304; Williams et al.J Hepatol. (2000) 32:754-761; Ohnishi et al. J Mol Cell Cardiol. (1998)30:2411-22; Lasky et al. Am J Physiol. (1998) 275: L365-371; Pan et al.Eur Respir J. (2001) 17:1220-1227; and Allen et al. Am J Respir Cell MolBiol. (1999) 21:693-700) Thus, CTGF represents a valid therapeutictarget in disorders, such as those described above.

Given the prevalence and severity of CTGF-associated diseases anddisorders, there is clearly a need for improved methods of treatmentthat can modulate CTGF expression. Antisense oligonucleotides representexceptional therapeutic agents based on their target specificity. Thepresent invention discloses antisense oligonucleotide compositions andmethods of use for modulating CTGF expression that can meet thesetherapeutic needs.

SUMMARY OF THE INVENTION

The present invention is directed to synthetic, i.e., non-naturallyoccurring, antisense oligonucleotides that are complementary to nucleicacids encoding human CTGF and modulate CTGF mRNA expression. Thedisclosed antisense oligonucleotides are useful for decreasing orinhibiting cellular CTGF mRNA and CTGF protein expression. Additionally,pharmaceutical and other compositions comprising the antisenseoligonucleotides of the invention are provided as well as methods oftheir use for the prevention or treatment of CTGF-associated healthconditions or diseases. Regions of the human CTGF mRNA that arehypersensitive to inhibition by antisense oligonucleotides are alsodisclosed.

In one aspect of the invention, a compound is provided comprising asynthetic oligonucleotide 8 to 50 nucleotides in length where at least acontiguous 8 nucleotide sequence of the oligonucleotide is complementaryto a region from nucleotides 567 to 588, 859 to 880, 981 to 1002, 989 to1010, 1061 to 1089, 1132 to 1153, 1179 to 1216, 1211 to 1232, 1329 to1350, 1623 to 1656, 1651 to 1672, 1750 to 1774, 1759 to 1786 or 1793 to1820 of SEQ ID NO: 1.

In one embodiment of the invention, at least an 8 nucleotide contiguoussequence of the oligonucleotide is complementary to a region fromnucleotides 567 to 588 of SEQ ID NO: 1.

In a further embodiment, at least an 8 nucleotide contiguous sequence ofoligonucleotide is complementary to a region from nucleotides 859 to 880of SEQ ID NO: 1.

In another embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 981 to1002 of SEQ ID NO: 1.

In one embodiment, at least an 8 nucleotide contiguous sequence of theoligonucleotide is complementary to a region from nucleotides 989 to1010 of SEQ ID NO: 1.

In another embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1061to 1089 of SEQ ID NO: 1.

In a further embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1132to 1153 of SEQ ID NO: 1.

In one embodiment, at least an 8 nucleotide contiguous sequence of theoligonucleotide is complementary to a region from nucleotides 1179 to1216 of SEQ ID NO: 1.

In another embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1211to 1232 of SEQ ID NO: 1.

In a further embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1329to 1350 of SEQ ID NO: 1.

In a one embodiment, at least an 8 nucleotide contiguous sequence of theoligonucleotide is complementary to a region from nucleotides 1623 to1656 of SEQ ID NO: 1.

In another embodiment, at least, an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1651to 1672 of SEQ ID NO: 1.

In a further embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1750to 1774 of SEQ ID NO: 1.

In a one embodiment, at least an 8 nucleotide contiguous sequence of theoligonucleotide is complementary to a region from nucleotides 1759 to1786 of SEQ ID NO: 1.

In another embodiment, at least an 8 nucleotide contiguous sequence ofthe oligonucleotide is complementary to a region from nucleotides 1793to 1820 of SEQ ID NO: 1.

In one aspect of the invention, compound is provided comprising asynthetic oligonucleotide 8 to 50 nucleotides in length, wherein atleast a contiguous 8 nucleotide sequence of the oligonucleotide iscomplementary to a region selected from the group of regions consistingof SEQ ID NO: 318 to SEQ ID NO: 340.

In a further aspect of the invention, a synthetic oligonucleotide isprovided selected from the group consisting of SEQ ID NO: 49, SEQ ID NO:56, SEQ ID NO: 62, SEQ ID NO: 182, SEQ ID NO: 66, SEQ ID NO: 183, SEQ IDNO: 184, SEQ ID NO: 71, SEQ ID NO: 187, SEQ ID NO: 75, SEQ ID NO: 76,SEQ ID NO: 77, SEQ ID NO: 192, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 193, SEQID NO: 81, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 82, SEQ ID NO:197, SEQ ID NO: 83, SEQ ID NO: 259, SEQ ID NO: 84, SEQ ID NO: 200, SEQID NO: 87, SEQ ID NO: 262, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:204, SEQ ID NO: 88, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 263, SEQID NO: 265, SEQ ID NO: 207, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 93; SEQ IDNO: 215, SEQ ID NO: 217, SEQ ID NO: 99, SEQ ID NO: 218, SEQ ID NO: 219,SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 100, SEQ ID NO: 222, SEQ IDNO: 223, SEQ ID NO: 101, SEQ ID NO: 270, SEQ ID NO: 103, SEQ ID NO: 271,SEQ ID NO: 226, SEQ ID NO: 104, SEQ ID NO: 227, SEQ ID NO: 272, SEQ IDNO: 105, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274,SEQ ID NO: 275, SEQ ID NO: 232, SEQ ID NO: 106, SEQ ID NO: 233, SEQ IDNO: 276, SEQ ID NO: 279, SEQ ID NO: 108, SEQ ID NO: 281, SEQ ID NO: 109,SEQ ID NO: 282, SEQ ID NO: 234, SEQ ID NO: 111, SEQ ID NO: 235, SEQ IDNO: 284, SEQ ID NO: 118, SEQ ID NO: 286, SEQ ID NO: 289, SEQ ID NO: 299,SEQ ID NO: 146, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 245, SEQ IDNO: 147, SEQ ID NO: 246, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 306,SEQ ID NO: 307, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 149, SEQ IDNO: 150, SEQ ID NO: 251, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311,SEQ ID NO: 312 and SEQ ID NO: 170.

In one embodiment, the oligonucleotide has at least one internucleosidelinkage, at least one sugar moiety, or at least one nucleobase that ismodified. In a further embodiment, the modified internucleoside linkageis a phosphothioate internucleoside linkage. In a still furtherembodiment, all of the internucleoside linkages of the oligonucleotideare phosphothioate internucleoside linkages.

In another embodiment, at least one modified sugar moiety of theoligonucleotide is a bicyclic sugar. In a further embodiment, themodified sugar comprises a 2′-O,4′-C-methylene bridge.

In one embodiment, at least one modified nucleobase of theoligonucleotide is a 5-methylcytosine.

In one aspect of the invention, a method for reducing the expression ofhuman CTGF mRNA is provided that comprises administering an effectiveamount of an oligonucleotide of the invention to a subject in needthereof, thereby reducing the expression of human CTGF mRNA.

In another aspect of the invention, a method of treating aCTGF-associated disorder is provided that comprises administering aneffective amount of an oligonucleotide of the invention to a subject inneed thereof, thereby treating the CTGT-associated disorder. In someembodiments, the CTGF-associated disease, condition or disorder isselected from the group consisting of dermal fibrosis, liver fibrosis,pulmonary fibrosis, renal fibrosis, cardiac fibrosis, ocular fibrosis,scleroderma, surgical scars and adhesions, scars from wounds, scars fromburns, restenosis, glomerular sclerosis, osteoarthritis and cancer. Infurther embodiments, the cancer is selected from the group consisting ofacute lymphoblastic leukemia, dermatofibromas, breast cancer, breastcarcinoma desmoplasia, angiolipoma, angioleiomyoma, desmoplastic cancer,prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer,gastrointestinal cancer, and liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleotide sequence of the entire spliced human CTGFmRNA sequence (NCBI Reference Sequence NM_(—)001901; SEQ ID NO: 1),wherein thymidine is substituted for uridine. The 2358-base sequenceincludes five exons: bases 1-272, 273-495, 406-747, 748-959 and960-2344, respectively with the coding sequences from bases 207-1256.

FIG. 2 is a histogram that shows the distribution of Round 1 antisenseoligonucleotides based on the percent inhibition of CTGF mRNA expression(knock down) compared to vehicle-treated control. A small shoulder isvisible on the right side of the expected bell-shaped curve of a normal(Gaussian) distribution indicating the presence of a larger thanexpected sub-population of potent antisense oligonucleotides that inducesignificant reduction in CTGF mRNA expression.

FIG. 3 is a graph of the Round 1 antisense oligonucleotides arranged asthe percent inhibition of CTGF mRNA expression induced by an individualantisense oligonucleotide (y-axis) versus its respective ranking(x-axis) for CTGF mRNA inhibitory activity. An inflection point isapparent at approximately 50% inhibition that indicates a larger thanexpected population of potent oligonucleotides.

FIG. 4 is a histogram that shows the distribution of Round 2 antisenseoligonucleotides based on the percent inhibition of CTGF mRNA expression(knock down) compared to vehicle-treated control. The distribution isshifted to the right compared to the Round 1 distribution shown in FIG.2 indicating that the population of oligonucleotides is substantiallyenriched for oligonucleotides that are potent inhibitors of CTGF mRNAactivity, i.e., inhibit at least 50% of the CTGF mRNA expressioncompared to vehicle-treated cells. The Round 2 antisenseoligonucleotides were designed to explore the sensitivity to antisenseoligonucleotide mediated inhibition of mRNA expression of regions ofmRNA immediately adjacent to and overlapping with those that arecomplementary to “seed” oligonucleotides from Round 1. These seedoligonucleotides induced at least 60% inhibition of CTGF mRNAexpression. The results in Round 2 demonstrate that many of the mRNAsequences that are adjacent to and overlapping with those that arecomplementary to the seed oligonucleotides are also very sensitive toantisense oligonucleotide mediated inhibition of activity. Thissuggested that there are regions of mRNA that are hypersensitive toantisense oligonucleotide mediated inhibition of mRNA expression, i.e.regions in which hybridization of a complementary antisenseoligonucleotide results in at least a 50% reduction in human CTGF mRNAexpression.

FIG. 5 is a histogram that shows the distribution of Round 3 antisenseoligonucleotides based on percent inhibition of CTGF mRNA expression(knock down) compared to vehicle-treated control. Like the Round 2antisense oligonucleotides, the Round 3 antisense oligonucleotides aresubstantially enriched for oligonucleotides that are potent inhibitorsof CTGF mRNA activity. The oligonucleotides synthesized in Round 3 weredesigned to test the boundaries of hypersensitive regions identified inRound 1 and Round 2.

FIGS. 6A-6E illustrate the distribution of the nucleotide sequences thatare complementary to Round 1-3 antisense oligonucleotides across themature human CTGF mRNA sequence, SEQ ID NO 1. (National Center forBiotechnology Information (NCBI) Reference Sequence NM_(—)001901)Additionally, these figures also show the degree of antisense mediatedknockdown of mRNA expression produced by each antisense oligonucleotide.Thus the position of each symbol (line) along the x-axis corresponds tothe complementary 20-nucleotide target site for each respectiveantisense oligonucleotide, while its position along the y-axiscorresponds to the level of CTGF mRNA expression after transfection ofthat oligonucleotide as a percent of vehicle-treated control, witholigonucleotides located below the dashed 50% line producing greaterthan 50% mRNA knockdown. FIG. 6A represents nucleotides 1-450 of humanCTGF mRNA and illustrates exon 1 and a portion of exon 2. FIG. 6Brepresents nucleotides 450-900 of human CTGF mRNA and illustrates exon 3and portions of exon 2 and exon 4. FIG. 6C represents nucleotides900-1350 of human CTGF mRNA and illustrate exon 5 and portions of exon 4and the 3′ UTR. FIGS. 6D and 6E represent nucleotides 1375-1825 and1825-2275, respectively, of human CTGF mRNA and illustrate the remainingportions of the 3′ UTR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises synthetic antisense oligonucleotidesthat modulate the expression of human CTGF mRNA. These antisenseoligonucleotides include isolated nucleic acids, nucleic acid mimetics,and combinations thereof. Also disclosed are regions of human CTGF mRNAthat are hypersensitive to antisense oligonucleotide mediated inhibitionof mRNA expression. Antisense oligonucleotides that modulate theexpression of CTGF mRNA are useful in situations where modulation ofCTGF mRNA expression represents an important intervention point in thetreatment of a CTGF-associated disease, condition of disorder.Modulation includes up-regulation or down-regulation of thetranscription rate of mRNA, rate or fidelity of splicing of pre-mRNA,total cellular mRNA level, rate of mRNA translation, and/or totalcellular, tissue or organ CTGF protein levels. Down-regulation of theexpression of CTGF mRNA can result in a decrease in or cessation ofcellular growth, a decrease in cellular replication, a decrease incellular motility, a decrease in cellular metabolism and/or theinduction of apoptosis. Additionally, down-regulation of expression ofCTGF mRNA can also result in alleviation of symptoms and improvement,cure or prevention of CTCF-associated diseases, conditions or disorders.

Definitions

The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, all nucleic acidsequences disclosed herein are expressed in the 5′-3′ direction.Additionally, unless otherwise indicated, a particular nucleic acidsequence in addition to explicitly indicating the disclosed sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions), alleles, and complementarysequences.

The term “oligonucleotide” and “oligomeric nucleic acid” refer tooligomers or polymers of ribonucleic acid (RNA), deoxyribonucleic acid(DNA), mimetics or analogs of RNA or DNA, or combinations thereof.Oligonucleotides are molecules formed by the covalent linkage of two ormore nucleotides or their analogs. Herein, a single nucleotide may alsobe referred to as a monomer or unit. The oligonucleotides of theinvention comprise contiguous nucleotide sequences between 8 to 50nucleotides in length. In some embodiments, the contiguous nucleotidesequences are at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48nucleotides in length. In other embodiments, the contiguous nucleotidesequences are not more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or50 nucleotides in length. In further embodiments, the contiguousnucleotide sequences are between 8 and 40, between 8 and 30, between 8and 24, between 8 and 20, between 10 and 40, between 10 and 30, between10 and 24, between 10 and 20, between 12 and 40, between 12 and 30,between 12 and 24, between 12 and 20, between 13 and 30, between 13 and24, between 13 and 20, between 16 and 24, or between 16 and 20nucleotides in length. Unless otherwise indicated, all oligonucleotidesequences disclosed herein are expressed in the 5′-3′ direction.

Oligonucleotides of the invention are linear molecules or aresynthesized as linear molecules. Preferably, the oligonucleotides do notform duplexes between sequences within individual molecules, in otherwords, the oligonucleotides are not substantially self-complementary. Insome embodiments, the oligonucleotides are essential not doubledstranded, i.e., not short hairpin RNA. In other embodiments, theoligonucleotides are single stranded. In some embodiments, theoligonucleotides are not small interfering RNAs (siRNAs). In otherembodiments, the oligonucleotides of the invention are not ribozymes,external guide sequence (EGS) oligonucleotides (oligozymes), or othershort catalytic RNAs.

The terms “small interfering RNA” or “siRNA” refer to single- ordouble-stranded RNA, molecules that induce the RNA-interference pathwayand act in concert with host proteins, e.g., RNA induced silencingcomplex (RISC) to degrade mRNA in a sequence-dependent fashion.

The terms “complementary” and “complementarity” refer to conventionalWatson-Crick base-pairing of nucleic acids. For example, in DNAcomplementarity, guanine forms a base pair with cytosine and adenineforms a base pair with thymine, whereas in RNA complementarity, guanineforms a base pair with cytosine, but adenine forms a base pair withuracil in place of thymine. An oligonucleotide is complementary to a RNAor DNA sequence when the nucleotides of the oligonucleotide are capableof forming hydrogen bonds with a sufficient number of nucleotides in thecorresponding RNA or DNA sequence to allow the oligonucleotide tohybridize with the RNA or DNA sequence. In some embodiments, theantisense oligonucleotides of the invention have perfect complementarityto human CTGF mRNA, i.e., no mismatches.

As used herein, the terms “antisense oligonucleotide” and “ASO” refer toan oligomeric nucleic acid that is capable of hybridizing with itscomplementary target nucleic acid sequence resulting in the modulationof the normal function of the target nucleic acid sequence. In someembodiments, the modulation of function is the interference in functionof DNA, typically resulting in decreased replication and/ortranscription of a target DNA. In other embodiments, the modulation offunction is the interference in function of RNA, typically resulting inimpaired splicing of transcribed RNA (pre-mRNA) to yield mature mRNAspecies, reduced RNA stability, decreased translocation of the targetmRNA to the site of protein translation and impaired translation ofprotein from mature mRNA. In other embodiments, the modulation offunction is the reduction in cellular target mRNA (CTGF) number orcellular content of target mRNA (CTGF). In some embodiments, themodulation of function is the down-regulation or knockdown of geneexpression. In other embodiments, the modulation of function is areduction in protein expression or cellular protein content. In furtherembodiments, the modulation of function is a phenotypic changeassociated with the reduction of CTGF including a reversion to a normalphenotype. In some embodiments, the change in phenotype includes achange in a cell's proliferation rate, migration rate, metastaticpotential, apoptosis rate, or sensitivity to chemotherapy agents,biologic agents or radiation. In other embodiments, the change inphenotype includes a change in the rate of tissue remodeling ordeposition of extracellular matrix. In some embodiments, a reduction inCTGF is associated with the reduction or alleviation of a symptom of aCTGF-associated disease, condition or disorder.

The antisense oligonucleotides provided herein may be defined by aparticular nucleotide sequence or SEQ ID NO. As used herein, anantisense oligonucleotide is identical to a sequence disclosed herein ifit has the same nucleotide base pairing ability. For example, a RNAsequence that contains uracil in place of thymidine in a disclosed DNAsequence would be considered identical to the DNA sequence since bothuracil and thymidine pair with adenine. Shortened and lengthenedversions of the antisense oligonucleotides described herein are alsocontemplated.

The antisense oligonucleotides of the invention effectively inhibit CTGFmRNA expression by at least 10%, 20%, 30%, 40%, 50%, 55% 60%, 65%, 70%,75%, 80%, 90% or 95% of that seen with vehicle treated controls i.e.,cells exposed only to the transfection agent and the PBS vehicle, butnot an antisense oligonucleotide. For example, the ASO SEQ ID NO: 246inhibited CTGF mRNA expression by 65% over that seen with thetransfection agent in PBS. In other embodiments, the administration ofan antisense oligonucleotide target to a CTGF nucleic acid results inthe reduction of CTGF mRNA expression of not more than about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95% of that seen with vehicle-treated controls. In still otherembodiments, the administration of an antisense oligonucleotide targetto a CTGF nucleic acid results in the reduction of CTGF mRNA expressionbetween about 10% and about 90%, about 20% and about 80%, about 30% andabout 70%, about 40% and about 60%, about 40% and about 70%, and about40% and about 80% of the expression level of vehicle treated controls.

The modulation of the CTGF mRNA or protein expression level can bedetermined using any of the standard molecular biology techniques knownto the art. For instance, mRNA levels can be determined using northernblotting or quantitative RT-PCR, while protein levels can be measuredusing ELISA, SDS-PAGE followed by western blotting or by massspectrometry.

As used herein, the terms “potent antisense oligonucleotide” or “potentASO” refer to an antisense oligonucleotide that has a surprisinglystrong inhibitory effect on CTGF mRNA expression. Potent antisenseoligonucleotides reduce the expression of human CTGF mRNA or protein byat least 50% compared to the expression level of vehicle-treatedcontrols. Potent antisense oligonucleotides of the invention comprise asequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO:176, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 182, SEQ ID NO: 66, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 71,SEQ ID NO: 187, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:192, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 253, SEQ ID NO: 254, SEQID NO: 255, SEQ ID NO: 256, SEQ ID NO: 193, SEQ ID NO: 81, SEQ ID NO:195, SEQ ID NO: 196, SEQ ID NO: 82, SEQ ID NO: 197, SEQ ID NO: 83, SEQID NO: 259, SEQ ID NO: 84, SEQ ID NO: 200, SEQ ID NO: 87, SEQ ID NO:262, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 204, SEQ ID NO: 88, SEQID NO: 205, SEQ ID NO: 206, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO:207, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 212, SEQ IDNO: 213, SEQ ID NO: 214; SEQ ID NO: 93; SEQ ID NO: 215, SEQ ID NO: 217,SEQ ID NO: 99, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ IDNO: 221, SEQ ID NO: 100, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 101,SEQ ID NO: 270, SEQ ID NO: 103, SEQ ID NO: 271, SEQ ID NO: 226, SEQ IDNO: 104, SEQ ID NO: 227, SEQ ID NO: 272, SEQ ID NO: 105, SEQ ID NO: 230,SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ IDNO: 232, SEQ ID NO: 106, SEQ ID NO: 233, SEQ ID NO: 276, SEQ ID NO: 279,SEQ ID NO: 108, SEQ ID NO: 281, SEQ ID NO: 109, SEQ ID NO: 282, SEQ IDNO: 234, SEQ ID NO: 111, SEQ ID NO: 235, SEQ ID NO: 284, SEQ ID NO: 118,SEQ ID NO: 286, SEQ ID NO: 130, SEQ ID NO: 287, SEQ ID NO: 236, SEQ IDNO: 288, SEQ ID NO: 237, SEQ ID NO: 132, SEQ ID NO: 238, SEQ ID NO: 133,SEQ ID NO: 289, SEQ ID NO: 135, SEQ ID NO: 291, SEQ ID NO: 292, SEQ IDNO: 293, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 139, SEQ ID NO: 241,SEQ ID NO: 242, SEQ ID NO: 296, SEQ ID NO: 243, SEQ ID NO: 141, SEQ IDNO: 289, SEQ ID NO: 299, SEQ ID NO: 146, SEQ ID NO: 300, SEQ ID NO: 302,SEQ ID NO: 245, SEQ ID NO: 147, SEQ ID NO: 246, SEQ ID NO: 303, SEQ IDNO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 247, SEQ ID NO: 248,SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 251, SEQ ID NO: 309, SEQ IDNO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 163 and SEQ ID NO:170

In some embodiments, the potent antisense oligonucleotides reduce theexpression of human CTGF mRNA or protein by at least 55% compared to theexpression level of vehicle-treated controls, i.e., SEQ ID NO: 43, SEQID NO: 176, SEQ ID NO: 49, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 182,SEQ ID NO: 66, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 71, SEQ ID NO:187, SEQ ID NO: 77, SEQ ID NO: 192, SEQ ID NO: 80, SEQ ID NO: 253, SEQID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 193, SEQ ID NO:81, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 82, SEQ ID NO: 197, SEQID NO: 83, SEQ ID NO: 259, SEQ ID NO: 84, SEQ ID NO: 200, SEQ ID NO: 87,SEQ ID NO: 262, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 204, SEQ IDNO: 88, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 90,SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:214, SEQ ID NO: 93, SEQ ID NO: 215, SEQ ID NO: 99, SEQ ID NO: 218, SEQID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 100, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 226, SEQID NO: 104, SEQ ID NO: 227, SEQ ID NO: 105, SEQ ID NO: 230, SEQ ID NO:231, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 232, SEQID NO: 106, SEQ ID NO: 233, SEQ ID NO: 279, SEQ ID NO: 108, SEQ ID NO:281, SEQ ID NO: 109, SEQ ID NO: 282, SEQ ID NO: 234, SEQ ID NO: 111, SEQID NO: 235, SEQ ID NO: 284, SEQ ID NO: 118, SEQ ID NO: 286, SEQ ID NO:130, SEQ ID NO: 287, SEQ ID NO: 236, SEQ ID NO: 288, SEQ ID NO: 237, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 289, SEQ ID NO: 135, SEQ ID NO:291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 239, SEQ ID NO: 240, SEQID NO: 139, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO:141, SEQ ID NO: 146, SEQ ID NO: 245, SEQ ID NO: 147, SEQ ID NO: 246, SEQID NO: 303, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO:247, SEQ ID NO: 248, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 251, SEQID NO: 309, and SEQ ID NO: 170.

In some embodiments, the potent antisense oligonucleotides reduce theexpression of human CTGF mRNA or protein by at least 60% compared to theexpression level of vehicle-treated controls, i.e., SEQ ID NO: 43, SEQID NO: 60; SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 183, SEQ ID NO: 192,SEQ ID NO: 80, SEQ ID NO: 253, SEQ ID NO: 193, SEQ ID NO: 81, SEQ ID NO:195, SEQ ID NO: 196, SEQ ID NO: 82, SEQ ID NO: 197, SEQ ID NO: 259, SEQID NO: 84, SEQ ID NO: 200, SEQ ID NO: 87, SEQ ID NO: 262, SEQ ID NO:203, SEQ ID NO: 204, SEQ ID NO: 204, SEQ ID NO: 88, SEQ ID NO: 205, SEQID NO: 206, SEQ ID NO: 207, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92,SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 93, SEQ ID NO: 215, SEQ IDNO: 99, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221,SEQ ID NO: 100, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 103, SEQ IDNO: 226, SEQ ID NO: 104, SEQ ID NO: 227, SEQ ID NO: 105, SEQ ID NO: 230,SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ IDNO: 232; SEQ ID NO: 106, SEQ ID NO: 233, SEQ ID NO: 281, SEQ ID NO: 234,SEQ ID NO: 111, SEQ ID NO: 235, SEQ ID NO: 284, SEQ ID NO: 236, SEQ IDNO: 237, SEQ ID NO: 132, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 292,SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 139, SEQ ID NO: 241, SEQ IDNO: 242, SEQ ID NO: 141, SEQ ID NO: 245, SEQ ID NO: 147, SEQ ID NO: 246,SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 247, SEQ ID NO: 149, SEQ IDNO: 150 and SEQ ID NO: 251.

In some embodiments, the potent antisense oligonucleotides reduce theexpression of human CTGF mRNA or protein by at least 65% compared to theexpression level of vehicle-treated controls, i.e., SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 66, SEQ ID NO: 183, SEQ ID NO: 253, SEQ ID NO:193, SEQ ID NO: 81, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 82, SEQID NO: 197, SEQ ID NO: 200, SEQ ID NO: 262, SEQ ID NO: 203, SEQ ID NO:204, SEQ ID NO: 204, SEQ ID NO: 88, SEQ ID NO: 205; SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 213,SEQ ID NO: 214, SEQ ID NO: 93, SEQ ID NO: 215, SEQ ID NO: 99, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 100, SEQID NO: 222, SEQ ID NO: 223, SEQ ID NO: 226, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 232, SEQID NO: 106, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 111, SEQ ID NO:235, SEQ ID NO: 284, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 289, SEQID NO: 240, SEQ ID NO: 139, SEQ ID NO: 242, SEQ ID NO: 246, SEQ ID NO:307, SEQ ID NO: 149 and SEQ ID NO: 150.

In some embodiments, the potent antisense reduce the expression of humanCTGF mRNA or protein by at least 70% compared to the expression level ofvehicle-treated controls, i.e., SEQ ID NO: 253, SEQ ID NO: 193, SEQ IDNO: 195, SEQ ID NO: 196, SEQ ID NO: 82, SEQ ID NO: 204, SEQ ID NO: 88,SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 214, SEQ ID NO: 215, SEQ IDNO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 274, SEQ ID NO: 232,SEQ ID NO: 234, SEQ ID NO: 235 and SEQ ID NO: 150.

In some embodiments, the potent antisense oligonucleotides reduce theexpression of human CTGF mRNA or protein by at least 75% compared to theexpression level of vehicle-treated controls, i.e., SEQ ID NO: 193, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 204, SEQ ID NO: 206 and SEQ IDNO: 222.

In some embodiments, the potent antisense oligonucleotides reduce theexpression of human CTGF mRNA or protein by at least 80% compared to theexpression level of vehicle-treated controls, i.e., SEQ ID NO: 204.

In other embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 43, SEQ ID NO: 176, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 60,SEQ ID NO: 62, SEQ ID NO: 182, SEQ ID NO: 66, SEQ ID NO: 183, SEQ ID NO:184, SEQ ID NO: 71, SEQ ID NO: 187, SEQ ID NO: 75; SEQ ID NO: 76; SEQ IDNO: 77, SEQ ID NO: 192, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 253,SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 193, SEQ IDNO: 194, SEQ ID NO: 81, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 82,SEQ ID NO: 197, SEQ ID NO: 83, SEQ ID NO: 259, SEQ ID NO: 84, SEQ ID NO:200, SEQ ID NO: 87, SEQ ID NO: 202, SEQ ID NO: 261, SEQ ID NO: 262, SEQID NO: 203, SEQ ID NO: 204, SEQ ID NO: 204, SEQ ID NO: 88, SEQ ID NO:205, SEQ ID NO: 206, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 207, SEQID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 212, SEQ ID NO: 213,SEQ ID NO: 214, SEQ ID NO: 93, SEQ ID NO: 215, SEQ ID NO: 217, SEQ IDNO: 99, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 267, SEQ ID NO: 220,SEQ ID NO: 221, SEQ ID NO: 100, SEQ ID NO: 222, SEQ ID NO: 223, SEQ IDNO: 101, SEQ ID NO: 270, SEQ ID NO: 103, SEQ ID NO: 271, SEQ ID NO: 226,SEQ ID NO: 104, SEQ ID NO: 227, SEQ ID NO: 272, SEQ ID NO: 228, SEQ IDNO: 105, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274,SEQ ID NO: 275, SEQ ID NO; 232, SEQ ID NO: 106, SEQ ID NO: 233, SEQ IDNO: 276, SEQ ID NO: 279, SEQ ID NO: 108, SEQ ID NO: 281, SEQ ID NO: 109,SEQ ID NO: 282, SEQ ID NO: 234, SEQ ID NO: 111, SEQ ID NO: 235, SEQ IDNO: 283, SEQ ID NO: 284, SEQ ID NO: 118, SEQ ID NO: 286, SEQ ID NO: 130,SEQ ID NO: 287, SEQ ID NO: 236, SEQ ID NO: 131, SEQ ID NO: 288, SEQ IDNO: 237, SEQ ID NO: 132, SEQ ID NO: 238, SEQ ID NO: 133, SEQ ID NO: 289,SEQ ID NO: 135, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ IDNO: 293, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 139, SEQ ID NO: 241,SEQ ID NO: 242, SEQ ID NO: 296, SEQ ID NO: 243, SEQ ID NO: 141, SEQ IDNO: 299, SEQ ID NO: 146, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 245,SEQ ID NO: 147, SEQ ID NO: 246, SEQ ID NO: 303, SEQ ID NO: 305, SEQ IDNO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 247, SEQ ID NO: 248,SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 249, SEQ ID NO: 150, SEQ IDNO: 251, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312,SEQ ID NO: 163 and SEQ ID NO: 170.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 43 and SEQ ID NO: 176.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 182, SEQ ID NO: 66, SEQ ID NO: 183 and SEQ ID NO: 184.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 71 and SEQ ID NO: 187.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 75 and SEQ ID NO: 76,

In other embodiments, the potent antisense oligonucleotides of theinvention comprise of a sequence selected from the group consisting ofSEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO:255, SEQ ID NO: 256, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 81, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 82 and SEQ ID NO: 197.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 83 and SEQ ID NO: 259.

In other embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group, consisting of SEQID NO: 84, and SEQ ID NO: 200.

In further embodiments, potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 87, SEQ ID NO: 202, SEQ ID NO: 261, SEQ ID NO: 203, SEQ ID NO:204, SEQ ID NO: 88, SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 263.

In certain embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 265, SEQ ID NO: 207 and SEQ ID NO: 90.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 93 and SEQ ID NO:215.

In other embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 217, SEQ ID NO: 99, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO:267, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 100, SEQ ID NO: 222 andSEQ ID NO: 223.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 101 and SEQ ID NO: 270.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 271, SEQ ID NO: 226, SEQ ID NO: 104, SEQ ID NO: 227, SEQ ID NO:272 and SEQ ID NO: 228.

In certain embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from group consisting of SEQ IDNO: 105, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274,SEQ ID NO: 275, SEQ ID NO: 232, SEQ ID NO: 106, SEQ ID NO: 233 and SEQID NO: 276.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 108, SEQ ID NO: 281 and SEQ ID NO: 109.

In other embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence select from group consisting of SEQ ID NO:282, SEQ ID NO: 234, SEQ ID NO: 111, SEQ ID NO: 235, SEQ ID NO: 283 andSEQ ID NO: 284.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise, a sequence selected from the group consisting of SEQID NO: 118 and SEQ ID NO: 286.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 130, SEQ ID NO: 287, SEQ ID NO: 236, SEQ ID NO: 131, SEQ ID NO:288, SEQ ID NO: 237, SEQ ID NO: 132, SEQ ID NO: 238, SEQ ID NO: 133 andSEQ ID NO: 289.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 291, SEQ ID NO: 292; SEQ ID NO: 293, SEQ ID NO: 239, SEQ ID NO:240, SEQ ID NO: 139, SEQ ID NO: 241, SEQ ID NO: 242.

In other embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 296, SEQ ID NO: 243 and SEQ ID NO: 141.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 299, SEQ ID NO: 146 and SEQ ID NO: 300.

In some embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 302, SEQ ID NO: 245, SEQ ID NO: 147, SEQ ID NO: 246 and SEQ IDNO: 303.

In other embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the group consisting of SEQID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO:247, SEQ ID NO: 248, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 249 andSEQ ID NO: 150.

In further embodiments, the potent antisense oligonucleotides of theinvention comprise a sequence selected from the consisting of SEQ ID NO:251, SEQ ID NO:309, SEQ ID NO: 310, SEQ ID NO: 311: and SEQ ID NO: 312.

It was further recognized, unexpectedly, that many of the targetsequences of the potent antisense oligonucleotides are clustered atspecific locations along the CTGF mRNA sequence as described below andshown in FIGS. 6A-6E and Table 2.

As used herein, the terms “modified” and “modification” when used in thecontext of the constituents of a nucleotide monomer, i.e., sugar,nucleobase and internucleoside linkage (backbone), refer to non-natural,changes to the chemical structure of these naturally occurringconstituents or the substitutions of these constituents withnon-naturally occurring ones, i.e., mimetics. For example, the“unmodified” or “naturally occurring” sugar ribose (RNA) can be modifiedby replacing the hydrogen at the 2-position of ribose with a methylgroup. See Monia, B. P. et al. J. Biol. Chem, 268: 14514-14522, 1993.Similarly, the naturally occurring internucleoside linkage is a 3′ to 5′phosphodiester linkage that can be modified by replacing one of thenon-bridging phosphate oxygen atoms with a sulfur atom to create aphosphorothioate linkage. See Geiser T. Ann N Y Acad Sci, 616: 173-183,1990.

When used in the context of an oligonucleotide, “modified” or“modification” refers to an oligonucleotide that incorporates one ormore modified sugar, nucleobase of internucleoside linkage. Modifiedoligonucleotide are structurally distinguishable, but functionallyinterchangeable with naturally occurring of synthetic unmodifiedoligonucleotides and usually have enhanced properties such as increasedresistance to degradation by exonucleases, or increased bindingaffinity.

It should be understood that the sequences set forth in SEQ ID NO formatare independent of any modification to a sugar moiety, aninternucleoside linkage, or a nucleobase. Therefore, an antisenseoligonucleotide defined by a SEQ ID NO may comprise, independently, oneor more modifications to a sugar moiety, an internucleoside linkage, ora nucleobase.

The term “target nucleic acid”, as used herein refers to the DNA or RNAencoding human CTGF protein or naturally occurring variants thereof. Theterm “naturally occurring variant thereof” refers to variants of thehuman CTGF nucleic acid sequence which exist naturally within thepopulation including any allelic variant, chromosomal translocation orduplication, or alternative splicing of human CTGF mRNA. In someembodiments, the RNA is mature mRNA, while in other embodiments the RNAis pre-mRNA pre-splice RNA. The potent ASOs according to the inventionare capable of hybridizing to specific “target sequences” within thetarget nucleic acid.

As used herein, the term “target sequence” refers to a human CTGF mRNAsequence within the target nucleic acid that is complementary to atleast an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguousnucleotide portion of an antisense oligonucleotide disclosed herein. Insome embodiments, the target sequences have perfect complementarity toan antisense oligonucleotide disclosed herein.

As used herein, the term “hypersensitive region.” refers to a region ofhuman CTGF mRNA sequence that is at least 21 nucleotides in lengthwherein hybridization of a complementary antisense oligonucleotide to aportion of the hypersensitive region results in at least a 50%inhibition of CTGF mRNA expression compared to vehicle-treated control.In some embodiments, the entire length of the antisense oligonucleotideis complementary to a portion of the hypersensitivity region. In otherembodiments, at least a contiguous 8 nucleotide sequence of theantisense oligonucleotide is complementary to a portion of thehypersensitive region. Hypersensitive regions of the invention are foundwithin the coding region and the 3′ UTR of human CTGF mRNA SEQ ID NO: 1and can be determined by inspection of Table 2. Additionally, thesequences of the empirically derived hypersensitive regions of humanCTGF mRNA are disclosed in Table 3 along with their respective SEQ IDNOs. A hypersensitive region may overlap with one or more otherhypersensitive regions.

In some embodiments, the potent antisense oligonucleotides of theinventions are complementary to a hypersensitive region from nucleotides567 to 588, 789 to 814, 859 to 880, 946 to 988, 981 to 1002, 989 to1010, 1006 to 1044, 1032 to 1055, 1061 to 1089, 1107 to 1145, 1132 to1153, 1164 to 1194, 1179 to 1216, 1211 to 1232, 1236 to 1265, 1329 to1350, 1514 to 1552, 1623 to 1656, 1651 to 1672, 1750 to 1774, 1759 to1786, 1771 to 1808 or 1793 to 1820 of SEQ ID NO: 1.

In other embodiments, the potent antisense oligonucleotides of theinvention are complementary, to a hypersensitive region from nucleotides567 to 588, 859 to 880, 981 to 1002, 989 to 1010, 1061 to 1089, 1132 to1153, 1179 to 1216, 1211 to 1232, 1329 to 1350, 1623 to 1656, 1651 to1672, 1750 to 1774, 1759 to 1786, or 1793 to 1820 of SEQ ID NO: 1.

In further embodiments, the potent antisense oligonucleotides of theinvention are complementary to a hypersensitive region from nucleotides859 to 880, 981 to 1002, 989 to 1010, 1061 to 1089, 1132 to 1153, 1179to 1216, 1211 to 1232, 1329 to 1350, 1750 to 1774, 1759 to 1786, or 1793to 1820 of SEQ ID NO: 1.

As can be seen in Table 2, certain sub-regions of the hypersensitiveregions of human CTGF mRNA when targeted by antisense oligonucleotideexhibit at least 55%, 60%, 65%, 70%, 75% or 80% inhibition of CTGF mRNAexpression. For example, the hypersensitive region from nucleotide 1236to 1265, SEQ ID NO: 332, has one sub-region that exhibits at least 65%inhibition; the sub-region from nucleotide 1238 to nucleotide 1261.

The terms “disorders” and “diseases” and “conditions” are usedinclusively and refer to any condition deviating from normal.

As used herein, “CTGF-associated disorders, conditions and diseases”refer to disorders, conditions and diseases associated with abnormal,dysregulated, especially increased expression of CTGF, or disorders,conditions and diseases that are affected by CTGF in terms of theirdevelopment, maintenance or progression. Abnormal expression of CTGF isassociated with hyperproliferative disorders that affect individualtissues, organs or multiple organs, including, but not limited to,angiolipoma, angioleiomyoma, dermatofibromas, and cancers, includingacute lymphoblastic leukemia, multiple myeloma, breast cancer,colorectal cancer, gastric cancer, gastrointestinal cancer, glioma andglioblastoma, head and neck cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cancer, rhabdomyosarcoma, desmoplasia,fibrosarcoma and metastases thereof.

CTGF-associated disorders also include fibrotic disorders that affectindividual tissues, organs or multiple organs, as in the case ofsystemic sclerosis. Fibrotic disorders include, for example, cardiacfibrosis, including cardiac reactive fibrosis or cardiac remodelingfollowing myocardial infarction or congestive heart failure;atherosclerosis, pulmonary fibrosis, including idiopathic pulmonaryfibrosis and asthma, joint fibrosis, fibrosis associated with dialysisincluding peritoneal dialysis; kidney fibrosis; liver fibrosis,interstitial fibrosis, scleroderma, skin fibrosis, keloids; fibrosisresulting from acute or repetitive traumas, including surgery i.e.,surgical adhesions such as abdominal and peritoneal adhesions,chemotherapy, radiation treatment, allograft rejection; or chronic andacute transplant rejection; fibrosis from hypertrophic scarring; andscarring from burns, wound healing and surgery.

Other CTGF-associated disorders include arthritis, including rheumatoidarthritis and osteoarthritis; Crohn's disease, inflammatory boweldisease; non-proliferative diabetic retinopathy, macular degeneration;nephropathies, including diabetic nephropathy, IgA-associatednephropathy, lupus kidney disease and nephropathy due to radiocontrastagents or other chemically induced renal toxicity; and conditionsassociated with chemical toxicity tubule destruction.

CTGF-associated disorders, conditions and diseases also refer toconditions and diseases that are associated with normal expressionlevels of CTGF, but the severity and progression of the condition ordisease is influenced by the CTGF level. For such conditions anddiseases, modulation of CTGF levels represents an intervention point forreducing the severity, or halting disease progression.

Sequences

The synthetic antisense oligonucleotides of the invention are 8 to 50nucleotides in length and correspond to complementary nucleotidesequences present in human CTGF mRNA. In some embodiments, the antisenseoligonucleotides are at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,or 48 nucleotides in length. In other embodiments, the antisenseoligonucleotides are not more than 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,or 50 nucleotides in length. In further embodiments, the antisenseoligonucleotides are between 8 and 40, between 8 and 30, between 8 and24, between 8 and 20, between 10 and 40, between, 10 and 30, between 10and 24, between 10 and 20, between 12 and 40, between 12 and 30, between12 and 24, between 12 and 20, between 13 and 30, between 13 and 24,between 13 and 20, between 16 and 24, or between 16 and 20 nucleotidesin length.

In some embodiments, the synthetic antisense oligonucleotides compriseat least a 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-merof 16-mer sequence of a 20-mer sequence selected from the groupconsisting of SEQ ID NO: 43; SEQ ID NO: 176, SEQ ID NO: 49, SEQ ID NO:56, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 182, SEQ ID NO: 66, SEQ IDNO: 183, SEQ ID NO: 184, SEQ ID NO: 71, SEQ ID NO: 187, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 192, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQID NO: 193, SEQ ID NO: 194, SEQ ID NO: 81, SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 82, SEQ ID NO: 197; SEQ ID NO: 83, SEQ ID NO: 259, SEQID NO: 84, SEQ ID NO: 200, SEQ ID NO: 87, SEQ ID NO: 202, SEQ ID NO:261, SEQ ID NO: 262, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 204, SEQID NO: 88, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 263, SEQ ID NO:265, SEQ ID NO: 207, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 93, SEQ ID NO: 215,SEQ ID NO: 217, SEQ ID NO: 99, SEQ ID NO: 218, SEQ ID NO: 219, SEQ IDNO: 267, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 100, SEQ ID NO: 222,SEQ ID NO: 223, SEQ ID NO: 101, SEQ ID NO: 270, SEQ ID NO: 103, SEQ IDNO: 271, SEQ ID NO: 226, SEQ ID NO: 104, SEQ ID NO: 227, SEQ ID NO: 272,SEQ ID NO: 228, SEQ ID NO: 105, SEQ ID NO: 230, SEQ ID NO: 231, SEQ IDNO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 232, SEQ TO NO: 106,SEQ ID NO: 233, SEQ ID NO: 276, SEQ ID NO: 279, SEQ ID NO: 108, SEQ IDNO: 281, SEQ ID NO: 109, SEQ ID NO: 282, SEQ ID NO: 234, SEQ ID NO: 111,SEQ ID NO: 235, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 118; SEQ IDNO: 286, SEQ ID NO: 130, SEQ ID NO: 287, SEQ ID NO: 236, SEQ ID NO: 131,SEQ ID NO: 288, SEQ ID NO: 237, SEQ ID NO: 132, SEQ ID NO: 238, SEQ IDNO: 133; SEQ ID NO: 289, SEQ ID NO: 135, SEQ ID NO: 290, SEQ ID NO: 291,SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 239, SEQ ID NO: 240, SEQ IDNO: 139, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 296, SEQ ID NO: 243,SEQ ID NO: 141, SEQ ID NO: 299, SEQ ID NO: 146, SEQ ID NO: 300, SEQ IDNO: 302, SEQ ID NO: 245, SEQ ID NO: 147, SEQ ID NO: 246, SEQ ID NO: 303,SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ IDNO: 247; SEQ ID NO: 248, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 249,SEQ ID NO: 150, SEQ ID NO: 251, SEQ ID NO: 309, SEQ ID NO: 310, SEQ IDNO: 311, SEQ ID NO: 312, SEQ ID NO: 163 and SEQ ID NO: 170.

In some embodiments, the synthetic antisense oligonucleotides comprise a20-mer sequence selected from the group consisting of SEQ ID NO: 43, SEQID NO: 176, SEQ ID NO: 49, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 62,SEQ ID NO: 182, SEQ ID NO: 66, SEQ ID NO: 183, SEQ ID NO: 184, SEQ IDNO: 71, SEQ ID NO: 187, SEQ ID NO: 75; SEQ ID NO: 76, SEQ ID NO: 77, SEQID NO: 192, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 253, SEQ ID NO:254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 81, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 82; SEQ ID NO:197, SEQ ID NO: 83, SEQ ID NO: 259, SEQ ID NO: 84, SEQ ID NO: 200, SEQID NO: 87, SEQ ID NO: 202, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO:203, SEQ ID NO: 204, SEQ ID NO: 204, SEQ ID NO: 88, SEQ ID NO: 205, SEQID NO: 206, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 207, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 212, SEQ NO: 213, SEQ IDNO: 214, SEQ ID NO: 93, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 99,SEQ ID NO: 218, SEQ ID NO: 219; SEQ ID NO: 267, SEQ ID NO: 220, SEQ IDNO: 221, SEQ ID NO: 100, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 101,SEQ ID NO: 270, SEQ ID NO: 103, SEQ ID NO: 271, SEQ ID NO: 226, SEQ IDNO: 104, SEQ ID NO: 227, SEQ ID NO: 272, SEQ ID NO: 228, SEQ ID NO: 105;SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 273, SEQ ID NO: 274, SEQ IDNO: 275, SEQ ID NO: 232, SEQ ID NO: 106, SEQ ID NO: 233, SEQ ID NO: 276,SEQ ID NO: 279, SEQ ID NO: 108; SEQ ID NO: 281, SEQ ID NO: 109, SEQ IDNO: 282, SEQ ID NO: 234, SEQ ID NO: 111, SEQ ID NO: 235, SEQ ID NO: 283,SEQ ID NO: 284, SEQ ID NO: 118, SEQ ID NO: 286, SEQ ID NO: 130, SEQ IDNO: 287, SEQ ID NO: 236, SEQ ID NO: 131, SEQ ID NO: 288, SEQ ID NO: 237,SEQ ID NO: 132, SEQ ID NO: 238, SEQ ID NO: 133, SEQ ID NO: 289, SEQ IDNO: 135, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293,SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 139, SEQ ID NO: 241, SEQ IDNO: 242, SEQ ID NO: 296, SEQ ID NO: 243, SEQ ID NO: 141, SEQ ID NO: 299,SEQ ID NO: 146, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 245, SEQ IDNO: 147, SEQ ID NO: 246, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 306,SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 247, SEQ ID NO: 248, SEQ IDNO: 148, SEQ ID NO: 149, SEQ ID NO: 249, SEQ ID NO: 150, SEQ ID NO: 251,SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ IDNO: 163 and SEQ ID NO: 170.

In some embodiments, the respective ends of a linear antisenseoligonucleotide are joined to form a circular structure. In someembodiments, the antisense oligonucleotides consist only of a singlestrand. In other embodiments, the antisense oligonucleotides furthercomprise a double strand, wherein the second strand is complementary toat least a portion of the first strand. In some embodiments, the secondoligonucleotide strand is not a guide strand or passenger strand of asiRNA construct.

In some embodiments, the antisense oligonucleotides further compriseheterologous nucleotide sequences, i.e., non-CTGF nucleotides sequences,at the 5′ and/or 3′ ends. In some embodiments, the heterologousnucleotide sequences comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 13, 14 or 15 nucleotides. When two or more heterologous,nucleotides are present, the added nucleotides may be at the 5′ end (5′addition), the 3′ end (3′ addition), or a combination thereof.

Oligonucleotide Composition

In some embodiments of the invention, the antisense oligonucleotidescomprise naturally-occurring nucleobases, sugars and covalentinternucleoside linkages, i.e., those found in naturally occurringnucleic acids. In other embodiments, the antisense oligonucleotidescomprise non-naturally occurring, i.e., modified nucleobases, sugarsand/or covalent internucleoside linkages. In further embodiments, theantisense oligonucleotides comprise a mixture of naturally occurring andnon-naturally occurring nucleobases, sugars and/or covalentinternucleoside linkages. Typically, oligonucleotides comprising whollyor partially of non-naturally occurring nucleobases, sugars or covalentinternucleoside linkages have improved characteristics overoligonucleotides composed wholly of naturally-occurring components. Forexample, oligonucleotides comprising non-natural covalentinternucleoside linkages usually have increased resistance todegradation from endonucleases or exonucleases resulting in increased invitro and in vivo half-lives compared to oligonucleotides consisting offonly naturally occurring covalent internucleoside linkages.Additionally, non-naturally occurring sugars can suitably enhance theaffinity of an antisense oligonucleotide for its target sequence.Alternatively, non-naturally occurring sugars may be useful in alteringimmune recognition of antisense oligonucleotides by pattern recognitionreceptors and other mechanisms, or alter other biologic pathways thatmay directly or indirectly affect the activity of an oligonucleotide,such as uptake, distribution, metabolism or efflux.

Non-naturally occurring covalent internucleoside linkages, i.e.,modified backbones, include those linkages that retain a phosphorus atomin the backbone and alto those that do not have a phosphorus atom in thebackbone. Numerous phosphorous containing modified oligonucleotidebackbones are known in the art and include, for example,phosphoramidites, phosphorodiamidate morpholinos, phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, and phosphinates. Insome embodiments, the modified antisense oligonucleotide backbones thatare without phosphorus atoms comprise short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. See Swayze E. and Bhat B. inAntisense Drug Technology Principles, Strategies, and Applications.2^(nd) Ed. CRC Press, Boca Rotan Fla., 2008 p. 144-182.

In further embodiments, the non-naturally occurring internucleosidelinkages are uncharged and in others, the linkages are achiral. In someembodiments, the non-naturally occurring internucleoside linkages areuncharged and achiral, e.g., peptide nucleic acids (PNAs).

In some embodiments, the modified sugar moiety is a sugar other thanribose or deoxyribose. In some embodiments, the sugar is arabinose,xylulose or hexose. In some embodiments, the sugar is substituted withone of the following at the 2′-position: OH, F; O-, S-, or N-alkyl; O-,S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10alkyl or C2 to C10 alkenyl and alkynyl. In some embodiments, themodifications include 2′-methoxy (2′-O—CH₃, 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. Similar modificationmay also be made at other positions on an oligonucleotide, particularlythe 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.

In some embodiments, the modified sugar is conformationally restricted.In further embodiments, the conformation restriction is the result ofthe sugar possessing a bicyclic moiety. In still further embodiments,the bicyclic moiety links the 2′-oxygen and the 3′ or 4′-carbon atoms.In some embodiments the linkages is a methylene (—CH₂-)n group bridgingthe 2′ oxygen atom and the 4′ carbon atom, wherein n is 1 or 2. Thistype of structural arrangement produces what are know as “locked nucleicacids” (LNAs). See Koshkin et al. Tetrahedron, 54, 3607-3630, 1998; andSingh et al., Chem. Commun, 455-456, 1998.

In some embodiments, the sugar is a sugar mimetic that isconformationally restricted resulting in a conformationally constrainedmonomer. In some embodiments, the sugar mimetic comprises a cyclohexylring that comprises one ring heteroatom and a bridge making the ringsystem bicyclic. See PCT/US2010/044549. In further embodiments, theantisense oligonucleotides comprise at least one nucleotide that has abicyclic sugar moiety or is otherwise conformationally restricted.

In some embodiments, the modified sugar moiety is a sugar mimetic thatcomprises a morpholino ring. In further embodiments, the phosphodiesterinternucleoside linkage is replaced with an uncharged phosphorodiamidatelinkage. See Summerton, Antisense Nucleic Acid Drug Dev., 7: 187-195,1997.

In some embodiments, both the phosphate groups and the sugar moietiesare replaced with a polyamide backbone comprised of repeatingN-(2-aminoethyl)-glycine units to which the nucleobases are attached viamethylene carbonyl linkers. The constructs are called peptide nucleicacids (PNAs). PNAs are achiral, uncharged and because of the peptidebonds, resistant to endo- and exonucleases. See Nielsen et al., Science,1991, 254, 1497-1500 and U.S. Pat. No. 5,539,082.

Antisense oligonucleotides of the invention include those comprisingentirely or partially of naturally occurring nucleobases. Naturallyoccurring nucleobases include adenine, guanine, thymine, cytosine,uracil, 5-methylcytidine, pseudouridine, dihydrouridine, inosine,ribothymidine, 7-methylguanosine, hypoxanthine and xanthine.

Antisense oligonucleotides of the invention also include thosecomprising entirely or partially of modified nucleobases(semi-synthetically or synthetically derived). Modified nucleobasesinclude 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,hypoxanthine, 2-aminoadenine, 2-methyladenine, 6-methyladenine,2-propyladenine, N6-adenine, N6-isopentenyladenine,2-methylthio-N6-isopentenyladenine, 2-methylguanine, 6-methylguanine,2-propylguanine, 1-methylguanine, 7-methylguanine, 2,2-dimethylguanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, dihydrouracil,5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyaceticacid methylester, uracil-5-oxyacetic acid,5-carboxymethylaminomethyl-2-thiouridine, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil,5-carboxymethylaminomethyluracil, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, 5-propynyl uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo-adenine,8-amino adenine, 8-thiol adenine, 8-thioalkyl adenine, 8-hydroxyadenine, 5-halo particularly 5-bromo, 5-trifluoromethyl uracil,3-methylcytosine, 5-methylcytosine, 5-trifluoromethyl cytosine,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine, 8-halo-guanine, 8-aminoguanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxy guanine,7-deazaadenine, 3-deazaguanine, 3-deazaadenine,beta-D-galactosylqueosine, beta-D-mannosylqueosine, inosine,1-methylinosine, 2,6-diaminopurine and queosine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2-(3H)-one), andphenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2-(3H)-one.See Herdewijn P, Antisense Nucleic Acid Drug Dev 10, 297-310, 2000; andSanghvi Y S, et al. Nucleic Acids Res, 21: 3197-3202, 1993.

In some embodiments, at least one nucleoside, i.e., a joined base andsugar, in an antisense oligonucleotide is modified, i.e., a nucleosidemimetic. In certain embodiments, the modified nucleoside comprises atetrahydropyran nucleoside, wherein a substituted tetrahydropyran ringreplaces the naturally occurring pentofuranose ring. SeePCT/US2010/022759 and PCT/US2010/023397. In other embodiments, thenucleoside mimetic comprises a 5′-substituent and a 2′-substituent. SeePCT/US2009/061913. In some embodiments, the nucleoside mimetic is asubstituted α-L-bicyclic nucleoside. See PCT/US2009/05813. In additionalembodiments, the nucleoside mimetic comprises a bicyclic sugar moiety.See PCT/US2009/039557. In further embodiments, the nucleoside mimeticcomprises a bis modified bicyclic nucleoside. See PCT/US2009/066863. Incertain embodiments, the nucleoside mimetic comprises a bicycliccyclohexyl ring wherein one of the ring carbons is replaced with aheteroatom. See PCT/US2009/03373. In still further embodiments, a 3′ or5′-terminal bicyclic nucleoside is attached covalently by a neutralinternucleoside linkage to the antisense oligonucleotide. SeePCT/US2009/039438. In other embodiments, the nucleoside mimetic is atricyclic nucleoside. See PCT/US2009/037686.

The aforementioned modifications may be incorporated uniformly across anentire antisense oligonucleotide, as specific regions or discretelocations within the oligonucleotide including at a single nucleotide.Incorporating these modifications can create chimeric or hybridantisense oligonucleotides wherein two or more chemically distinct areasexist, each made up of one or more nucleotides.

Chimeric oligonucleotides typically contain at least one region that ismodified to improve at least one property of the oligonucleotidecompared to the unmodified oligonucleotide. Improved properties includedfor instance increased resistance to nuclease degradation, increasedcellular uptake, increased binding affinity for a specific sequencewithin the target nucleic acid and/or increased inhibition of targetmRNA expression. Consequently, chimeric oligonucleotides are oftenpreferred over unmodified oligonucleotides. Chimeric oligonucleotides,include a particular subset known as “gapmers.” See Monia et al, J. BiolChem: 268: 14514-14522, 1993; Altman et al. Chimia, 50: 168-176, 1996;Seth et al. J Med Chem 52: 10-13, 2009; WO/US92/011339 andWO/1993/013121.

In some embodiments, the antisense oligonucleotide are gapmers that havea central “gap” region comprising 2′-deoxynucleotides flanked on bothsides by “wings” composed of 2′-methoxyethyl (2′-MOE) nucleotides. Insome embodiments, gapmers comprise phosphorothioate internucleoside(backbone) linkages throughout the oligonucleotide. In otherembodiments, the gapmers comprise phosphorothioate linkages in thecentral gap and phosphodiester linkages in the wings. In someembodiments, the gapmers comprise a sequence selected from the groupconsisting of SEQ ID NO: 43, SEQ ID NO: 176, SEQ ID NO: 49, SEQ ID NO:56, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 182, SEQ ID NO: 66, SEQ IDNO: 183, SEQ ID NO: 184, SEQ ID NO: 71, SEQ ID NO: 187, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 192, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255; SEQ ID NO: 256, SEQID NO: 193, SEQ ID NO: 194, SEQ NO: 81, SEQ ID NO: 195, SEQ ID NO: 196,SEQ ID NO: 82, SEQ ID NO: 197, SEQ ID NO: 83, SEQ ID NO: 259; SEQ ID NO:84, SEQ ID NO: 200, SEQ ID NO: 87, SEQ ID NO: 202; SEQ ID NO: 261, SEQID NO: 262, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 204, SEQ ID NO:88, SEQ ID NO: 205; SEQ ID NO: 206, SEQ ID NO: 263, SEQ ID NO: 265, SEQID NO: 207, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 212,SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 93, SEQ ID NO: 215, SEQ IDNO: 217, SEQ ID NO: 99, SEQ ID NO: 218, SEQ ID NO: 219; SEQ ID NO: 267,SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 100; SEQ ID NO: 222, SEQ IDNO: 223, SEQ ID NO: 101, SEQ ID NO: 270; SEQ ID NO: 103, SEQ ID NO: 271,SEQ ID NO: 226; SEQ ID NO: 104, SEQ ID NO: 227, SEQ ID NO: 272, SEQ IDNO: 228, SEQ ID NO: 105, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 273,SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 232, SEQ ID NO: 106, SEQ IDNO: 233, SEQ ID NO: 276, SEQ ID NO: 279, SEQ ID NO: 108, SEQ ID NO: 281,SEQ ID NO: 109, SEQ ID NO: 282; SEQ ID NO: 234, SEQ ID NO: 111, SEQ IDNO: 235, SEQ ID NO: 283; SEQ ID NO: 284, SEQ ID NO: 118; SEQ ID NO: 286,SEQ ID NO: 130, SEQ ID NO: 287, SEQ ID NO: 236, SEQ ID NO: 131, SEQ IDNO: 288, SEQ ID NO: 237, SEQ ID NO: 132, SEQ ID NO: 238, SEQ ID NO: 133,SEQ ID NO: 289, SEQ ID NO: 135, SEQ ID NO: 290, SEQ ID NO: 291, SEQ IDNO: 292, SEQ ID NO: 293, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 139,SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 296, SEQ ID NO: 243, SEQ IDNO: 141, SEQ ID NO: 299, SEQ ID NO: 146, SEQ ID NO: 300, SEQ ID NO: 302,SEQ ID NO: 245, SEQ ID NO: 147, SEQ ID NO: 246, SEQ ID NO: 303, SEQ IDNO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 247,SEQ ID NO: 248, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 249, SEQ IDNO: 150, SEQ ID NO: 251, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311,SEQ ID NO: 312, SEQ ID NO: 163 and SEQ ID NO: 170.

In other embodiments, the gapmers are complementary to a hypersensitiveregion selected from SEQ ID NO: 318 to SEQ ID NO: 340 (Table 3).

In further embodiments, particular gapmers are envisioned comprising (a)a gap segment comprising linked deoxynucleosides, (b) a 5′ wing segmentcomprising linked modified nucleosides; and (c) a 3′ wing segmentcomprising linked modified nucleosides, wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment andwherein each modified nucleoside within each wing segment comprises amodified sugar. In further embodiments, the modified sugar comprises a2′-O,4-C-methylene bridge. In still further embodiments, the gapmercomprises: (a) a gap segment comprising 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16 linked deoxynucleosides, (b) a 5′ wing segment comprising1, 2, 3, 4, 5, 6 or 7 linked modified nucleosides; and (c) a 3′ wingsegment comprising 1, 2, 3, 4, 5, 6 or 7 linked modified nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment; wherein each modified nucleoside within each wingsegment comprises of a modified sugar comprising a 2′-O,4′-C-methylenebridge; and wherein each internucleoside linkage is a phosphothioatelinkage. In a preferred embodiment, the antisense oligonucleotides havea 2-16-2, 3-14-3, 2-13-5 or 4-12-4, 5′ wing segment-gap-3′ wing segmentmotif. See PCT/US2009/054973.

In some embodiments, the antisense oligonucleotides further comprise anucleotide tail, wherein the nucleotides of the tail have phosphodiesterbackbones or other suitable backbones to impart increased solubility tothe antisense molecule, as disclosed in Toon et al. (British JHaematology, 96: 377-381, 1997). The use of a soluble tail can obviatethe need to use liposomes, for delivery allowing the antisense moleculeto be dissolved directly into a suitable aqueous medium.

In some embodiments, the antisense oligonucleotides further comprise aheterogeneous molecule covalently attached to the oligomer, with orwithout the use of a linker, also known as a crosslinker. In someembodiments, the heterogeneous molecule is a delivery or internalizationmoiety that enhances or assists the absorption, distribution and/orcellular uptake of the oligonucleotides. These moieties includepolyethylene glycols, cholesterols, phospholipids, cell-penetratingpeptides (CPPs), ligands to cell membrane receptors and antibodies. SeeManoharan M. in Antisense Drug Technology: Principles, Strategies andApplications, Crooke S T, ed. Marcel Dekker, New York, N.Y., 2001, p.391-470.

The disclosed antisense oligonucleotides may be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Life Technologies Corporation (Carlsbad,Calif.). Any other means for such synthesis known in the art mayalternatively be employed. Additionally, numerous service providers canbe contracted to prepare the disclosed compounds.

Applications

The antisense oligonucleotides of the invention may be utilized asresearch reagents including, for example, in biomedical scienceapplications and in the development of diagnostics or therapeuticsapplications. In biomedical research, such antisense oligonucleotidesmay be used to specifically inhibit the synthesis of human CTGF protein(typically by degrading CTGF mRNA of inhibiting the transcription ortranslation of CTGF mRNA) in cells and experimental animals tofacilitate the understanding of the role that CTGF may play underphysiological or pathological conditions.

In diagnostics, the antisense oligonucleotides may be used to detect andquantitate CTGF mRNA expression in cells and tissues by northernblotting, in-situ hybridization or similar techniques. For experimentaltherapeutics, an animal suspected of having a CTGF-associated disease ordisorder can be treated by administering antisense oligonucleotides inaccordance with this invention. Monitoring the response of the animalwould allow researchers to better understand the role of CTGF in diseaseetiology and progression. From this understanding, better diagnostic ortherapeutic treatment methods including antisense oligonucleotidetreatment methods can be developed.

The antisense oligonucleotides of the invention can be usedtherapeutically to treat CTGF-associated disorders, conditions anddiseases when administered alone or formulated as pharmaceuticalcompositions as described below. CTGF-associated disorders include, butare not limited to, hyperproliterative disorders including angiolipoma,angioleiomyoma, dermatofibromas, and cancers, including acutelymphoblastic leukemia, multiple myeloma, breast cancer, colorectalcancer, gastric cancer, gastrointestinal cancer, glioma andglioblastoma, head and neck cancer, liver cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cancer,rhabdomyosarcoma, desmoplasia, fibrosarcoma and metastases thereof.

CTGF-associated disorders also include fibrotic disorders that affectindividual tissues, organs or multiple organs, as in the case ofsystemic sclerosis. Fibrotic disorders include, for example, cardiacfibrosis, including cardiac reactive fibrosis or cardiac remodelingfollowing myocardial infarction or congestive heart-failure;atherosclerosis, pulmonary fibrosis, including idiopathic pulmonaryfibrosis and asthma; fibrosis associated with dialysis includingperitoneal dialysis; kidney fibrosis; liver fibrosis; interstitialfibrosis, scleroderma; skin-fibrosis including scars; keloids; andfibrosis resulting from acute or repetitive traumas, including surgery,chemotherapy, radiation treatment; allograft rejection; or chronic andacute transplant rejection.

Other CTGF-associated disorders include arthritis, including rheumatoidarthritis and osteoarthritis; Crohn's disease; inflammatory boweldisease; non-proliferative diabetic retinopathy, macular degeneration;nephropathies, including diabetic nephropathy, IgA-associatednephropathy, lupus kidney disease and nephropathy due to radiocontrastagents or other chemically induced renal toxicity; and conditionsassociated with chemical toxicity tubule destruction.

In some embodiments, methods are provided for reducing hypertropicscarring resulting from dermal wound healing in a subject in needthereof. In these methods, an antisense oligonucleotide is administeredin an effective amount to inhibit expression of CTGF, thereby reducingscarring from wound healing. In some embodiments, the wound healing ishealing at a wound selected from the group consisting of skin breakage,surgical incisions and burns. In further embodiments, the subject isadministered an antisense oligonucleotide before a surgical procedure isperformed. In other embodiments, methods are provided for reducingadhesions including those from surgery. In these methods, an antisenseoligonucleotide is administered in an effective amount to inhibitexpression of CTGF, thereby reducing the formation of adhesions.

In further embodiments, methods are provided for treating an individualprophylactically to reduce current or expected level of CTGF expressionthereby preventing, reducing, attenuating or delaying the onset orseverity of a future CTGF-associated disease, condition of disorder. Insome embodiments, the individual is predisposed or has an elevated riskof developing a CTGF-associated disease, condition or disorder basedupon, occupational, genetic, previous medical history or other factors.In some embodiments, the previous medical history includes, but is notlimited to, heart disease, diabetes, obesity or treatment withradiotherapy or chemotherapeutic agents.

Suitable routes of administration include parenteral (systemic) andloco-regional including topical administration. Parenteraladministration includes oral, intravenous, intramuscular; subcutaneous,intra-arterial, intra-articular and pulmonary administration.Loco-regional administration includes intraperitoneal, retroperitoneal,intradermal, epidermal, intralesional, ocular, intraocular,intrapleural, intrathecal, intraventricular, intravesical, intranasal,vaginal, bladder and buccal administration. It is understood thatappreciable systemic exposure can be achieved through loco-regionaladministration, if desired, through the use of appropriate formulations,administered quantities of ASOs and dosing schedules. The indication tobe treated and treatment intent dictate the route of administration tobe used and consequently the type of formulation.

Pharmaceutical Compositions

The present invention also includes pharmaceutical compositions andformulations that comprise the disclosed antisense oligonucleotides. Oneof skill in the art will recognize that the formulation employed willdepend in part on the intended route of administration. Variousformulations and drug delivery systems are available in the art. See,e.g., Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences,18th ed., Mack Publishing Co., and Hardman, Limbird and Gilman, eds(2001) The Pharmacological Basis of Therapeutics, McGraw-Hill, New York,N.Y.

Typically, the antisense oligonucleotides are present in pharmaceuticalcompositions and formulations as pharmaceutically acceptable salts,i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Pharmaceutically acceptable salts include base addition salts that areformed with metals, for example, sodium, potassium, magnesium or calciumcations, or as organic amines, for example, chloroprocaine, choline,diethanolamine or ethylenediamine. Pharmaceutically acceptable saltsalso include organic or inorganic acid salts of amines, for example,hydrochlorides, acetates or phosphates. Other suitable pharmaceuticallyacceptable salts are well known to those skilled in the art.

Compositions and formulations for injection include sterile aqueoussolutions or emulsions that may further contain buffers, diluents,carriers, preservatives, stabilizer and other excipients. Compositionsand formulations for oral administration include tablets, capsules, gelcapsules, dragees, powders, suspensions, emulsions, microemulsions orsolutions. Typically, the compositions and formulations for oraladministration further include binders, bulking agents, carriers,coloring agents, flavoring agents, surfactants, chelators, emulsifiersand other excipients. Preferred surfactants include fatty acids, estersof fatty acids, bile acids and their salts. Pharmaceutically acceptableexcipients are available in the art, and include those listed in variouspharmacopoeias. See, e.g., USP, JP, EP, and BP, and Handbook ofPharmaceutical Additives, ed. Ash; Synapse Information Resources, Inc.2002.

Compositions and formulations for topical administration includetransdermal patches, ointments, lotions, creams, gels, foams, sprays andliquids. The antisense oligonucleotides of the invention can bedelivered transdermally by iontophoretic delivery, electroporation,microneedle arrays or chemical penetration enhancers including lipidsand liposomes.

As used herein, the term “liposome” means a vesicle composed ofamphiphilic lipids arranged in a spherical bilayer or bilayers.Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior thatcontains the antisense oligonucleotides. Liposomes can protectencapsulated drugs in their internal compartments from metabolism anddegradation, increase drug accumulation at a target site, and reducedrug toxicity. See Lian T. and Ho, R, J. Y. J Pharma Sci, 90: 667-680,2001

Lipids and liposomes include neutral lipids, e.g., dioleoylphosphatidylethanolamine and distearolyphosphatidyl choline; negative lipids, e.g.,dimyristoylphosphatidyl glycerol and cationic lipids, e.g.,dioleoylphosphatidyl ethanolamine dioleyloxypropyltrimethyl ammoniumchloride.

For parenteral administration, liposomes may incorporate glycolipids orbe derivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) to enhance circulation lifetimes relative toliposomes lacking such specialized lipids or hydrophilic polymers. SeeUster P. S. et al. FEBS Letters, 1996, 386: 243-246. Additionally,liposomes can be targeted to specific cell types by coupling theliposome to antibodies, antibody fragments or ligands. See Yu B et al.Am Asso Pharma Sci, 11: 195-203, 2009

Preferred formulations for topical administration include those in whichthe antisense oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants.

The compositions of the invention may be provided in an instant release,controlled release or sustained release formulation with formulationingredients dependent on the administration route and treatment intent.

In some embodiments, pharmaceutical compositions contain two or moreantisense oligonucleotides, each targeted to the same hypersensitiveregion of CTGF mRNA. In some embodiments, the combination of two or moreantisense oligonucleotides produces at least an additive degree ofinhibition of CTGF mRNA expression. In other embodiments, thecombination of two or more antisense oligonucleotides produces asynergistic degree of inhibition of CTGF mRNA expression.

In other embodiments, pharmaceutical compositions contain two or moreantisense oligonucleotides, each targeted to a different hypersensitiveregion of CTGF mRNA. In some embodiments, the combination of two or moreantisense oligonucleotides produces at least an additive degree ofinhibition of CTGF mRNA expression. In other embodiments, thecombination of two or more antisense oligonucleotides produces asynergistic degree of inhibition of CTGF mRNA expression.

An additional benefit of administering pharmaceutical compositionscontaining at least two antisense oligonucleotides at a total dosagethat is equivalent to an effective dosage of a single antisenseoligonucleotide is that any sequence specific off-target effects causedby one particular antisense oligonucleotide will be diluted out orminimized through the use of the other antisense oligonucleotide whilemaintaining the desired efficacy.

In further embodiments, the pharmaceutical compositions contain at leastone antisense oligonucleotide and another pharmaceutical agent thatfunctions by a non-antisense mechanism, such as a small molecule drug,antibody or other immunotherapy agent. Examples of small molecule drugsinclude, but are not limited to, cancer chemotherapeutics,anti-inflammatory drugs, analgesics and anti-virals.

The antisense oligonucleotides of the invention can also be provided oradministered as prodrugs. As used herein, the term “prodrug” indicates atherapeutic agent that is prepared in an active form and followingadministration to an animal, including humans, the prodrug is convertedto the biologically active antisense oligonucleotide typically throughthe action of endogenous enzymes or changes in pH. In some embodiments,prodrug versions of the antisense oligonucleotides of the invention areprepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives. SeeTosquella G. et al. Nucleic Acids Res, 26: 2069-2074, 1998.

Antisense Oligonucleotide Dosages and Dosing Schedules

The pharmaceutical formulations of the present invention may bemanufactured or dispensed into single unit or multi-unit dosage formsusing techniques well known in the pharmaceutical industry. Single unitand multi-unit dosage forms, include, but are not limited to, tablets,capsules, gel capsules, tubes, suppositories, patches, vials andpre-filled syringes.

A therapeutically effective dose of an antisense oligonucleotidecomposition refers to an amount of antisense oligonucleotide thatameliorates or reduces the symptoms of a CTGF-associated condition. Atherapeutically effective dose also includes an amount of antisenseoligonucleotide that prevents the development, slows the progression orcures a CTGF-associated condition.

A therapeutically effective dose can be estimated initially using avariety of techniques well-known in the art. Initial doses used inanimal studies may be based on effective concentrations established incell culture assays. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from animal studies andcell culture assays.

Dosing is dependent on the severity of the disease and theresponsiveness of the disease to therapy among other factors. In someembodiments of the invention, the antisense oligonucleotide isadministered at a dose of at least about 0.001, 0.01, 0.05, 0.1, 0.5, 1,2, 3, 4, 5, 10 or 100 mg/kg of body weight. In other embodiments, theantisense oligonucleotide is administered at a dose of no more thanabout 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 50 or 100 mg/kg ofbody weight. Alternately, in some embodiments, the antisenseoligonucleotide is administered at a dose of at least about 0.01, 0.1,1, 10, 100 or 1000 mg/m² of body surface area (BSA). In otherembodiments, the antisense oligonucleotide is administered at a dose ofnot more than about 1, 10, 100, 1,000 or 5,000 mg/m² of BSA. Inparticular embodiments, for the prevention or reduction of hypertropicscarring, the antisense oligonucleotide is administered between 1.0μg/cm to 10 mg/cm of the wound length.

A patient can receive a single treatment or a course of treatmentlasting from two or more days, weeks, months or years. Alternately, apatient can be treated until a cure is affected of a diminution of thedisease state or symptoms is achieved. Following successful treatment,it may be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state, wherein the antisenseoligonucleotide administered in maintenance doses, ranging from at leastabout 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 50 or 100 mg perkg of body weight, daily, weekly, monthly or yearly as necessary.

Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosage may vary depending onthe relative potency of individual antisense oligonucleotides, and cangenerally be estimated based on an EC₅₀ found to be effective in an invitro or in vivo model. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body of a patient. Thepharmaceutical compositions of the invention can be administered at thesame time as one or more other pharmaceutical agents or at differenttimes.

For therapeutics, a mammal, preferably a human, having a disease,condition or disorder, or at risk for developing one, is treated bymodulating the expression of CTGF through administration of one or moreantisense oligonucleotides in accordance with this invention. Forexample, in some non-limiting embodiments, the treatment methodcomprises the step of administering to a mammal in need thereof, atherapeutically effect amount of an antisense oligonucleotide thattargets CTGF mRNA. In some embodiments, the antisense oligonucleotidesare administered before the administration of another treatment modalityor medical procedure, such as radiation therapy. In other embodiments,the antisense oligonucleotides are administered after an earliertreatment modality or procedure, for example, after surgery, for theprevention or reduction of scarring.

Kits

The antisense oligonucleotides and compositions of the present inventioncan be supplied to researchers and healthcare practitioners as kits.Typically, research kits include one or more containers holding one ormore CTGF antisense oligonucleotides and instructions for using theoligonucleotides for the purpose of modulating CTGF expression. Kits formedical use typically include one or more packs or dispenser devicescontaining one or more unit dosage forms of the antisenseoligonucleotide compositions and instructions for their administration.Such a pack or device may, for example, comprise metal or plastic foil,such as a blister pack; or plastic bottle and cap. Compositionscomprising an agent of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer such as a metal or plastic tube with cap and labeled fortreatment of an indicated condition.

The following examples serve only to illustrate and aid in theunderstanding of the invention and are not intended to limit theinvention as set forth in the claims which follow thereafter. Variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description. Such modifications are intended to fall withinthe scope of the appended claims. All references cited herein are herebyincorporated by references in their entirety.

EXAMPLES Example 1 In Silico Identification of AntisenseOligonucleotides

Antisense oligonucleotides are known to inhibit gene expression. In aneffort to identify novel, highly active antisense oligonucleotides tohuman CTGF, the entire spliced human CTGF mRNA sequence (NCBI ReferenceSequence NM_(—)001901; SEQ ID NO: 1; FIG. 1), including the 5′ UTR(untranslated region) and 3′UTR, was screened in silico. To facilitatethe screening process, the CTGF mRNA representing the coding sequencewas divided into consecutive, 10 nucleotide sequence windows, while the5′ UTR and 3′ UTR regions were divided into consecutive, 15 nucleotidesequence windows. Regions of low sequence of complexity and simpleinterspersed repeats were identified using RepeatMasker. 3.2.7 run onthe Crossmatch search engine (AFA Smit, Institute for Systems Biology).These regions were excluded from further analysis leaving a total of 181screening windows.

The nucleic acid sequences represented by each screening window servedas the starting point for in silico hybridization with a set of either10 complementary oligonucleotides (coding region) of 15 complementaryoligonucleotides (5′ UTR and 3′ UTR). Each oligonucleotide in a set was20 nucleotides in length with one portion of the oligonucleotidehybridizing by its 3′ end to its complementary nucleotide sequencewithin the screening window and the remaining portion of theoligonucleotide hybridizing to the mRNA sequence that was immediatelyadjacent to the window in the 3′ direction of the mRNA strand. Thesequence of CTGF mRNA that is complementary to a test oligonucleotidewas termed a “target sequence”.

Using a coding sequence screening window that contains a sequence of 10nucleotides as an example, the screening window will have ten, 20-merASOs that are each complementary to a portion of the human CTFG mRNAsequence represented by the screening window. The 3′ end of these ASOsmust hybridize to the CTGF mRNA beginning somewhere within the window.One ASO will hybridize to the complete 10 nucleotide screening windowsequence with the remaining 10 nucleotides of the antisenseoligonucleotide hybridizing to the CTGF mRNA that is contiguous to thewindow in the direction of the 3′ end of the mRNA strand. Anotherantisense oligonucleotide will hybridize to 9 nucleotides in thescreening window sequence and to 11 nucleotides in the CTGF mRNA that iscontiguous to the window in the direction of the 3′ end of the mRNAstrand. And so on, until the last oligonucleotide in the serieshybridizes to 1 nucleotide in the screening window and to 19 nucleotidesin the CTGF mRNA that is contiguous to the window in the direction ofthe 3′ end of the mRNA strand. In this way, a set of tenoligonucleotides was generated for every screening window representing a10 nucleotide portion of the CTGF mRNA coding region. Eacholigonucleotide in a set possesses perfect complementarity to its targetsequence of CTGF mRNA. Similarly, a set of 15 complementaryoligonucleotides was generated for every screening window representingthe 5′ UTR and 3′ UTR.

Next, each 20-mer oligonucleotide within an individual screening windowset was assigned a theoretical score based on the Gibbs free energiesfor inter- and intra-oligonucleotide and oligonucleotide-to-targetbase-pairing, where free energy values at 37° C. were estimated usingthe OligoWalk and OligoScreen, modules of RNA structure v4.6 (Mathews etal. Proc Natl Acad Sci USA 2004, 101:7287-92). The respective freeenergy Z-scores were multiplied by coefficients of correlation obtainedby Matveeva et al. for the thermodynamic properties and antisenseactivities of 1224 phosphorothioate ASOs (Matveeva et al. Nucleic AcidsRes 2003, 31:4989-94), and where weighted Z-scores were summed to obtainoligonucleotide-specific thermodynamic scores.

Thus, the thermodynamic score for each oligonucleotide was calculated as10×(0.16× the Z-score for the inter-oligonucleotide dimerization freeenergy+0.12×the Z-score for the intra-oligonucleotide self-pairing freeenergy−0.24×the Z-score for the free energy of oligonucleotide-to-targetmRNA duplex formation), where the Z-score for a given oligonucleotideand type of inter- or intra-molecular base-pairing was calculated as thedifference between its specific Gibbs free energy value (ΔG37) and meanfree energy for all 2325 possible 20-mer ASOs along the CTGF mRNAsequence, divided by the standard deviation (SD) of the free energiesfor all possible 20-mers, i.e., z=(ΔG37−means)/SD. The thermodynamicscores for all 2325 possible 20-mer ASOs ranged from −9.66 to 7.63(median=0.07, mean±SD=−0.22±2.43).

From these calculations, the oligonucleotide with the highest score(i.e., the greatest theoretical likelihood of possessing high antisenseactivity) was chosen from each set and was termed a “screeningoligonucleotide.” Discretionary allowances were made for high-scoringoligonucleotides that fell just outside a given screening window, sothat some sets contributed more than one oligonucleotide to the overallcollection of screening oligonucleotides, while other windowscontributed no oligonucleotides. A total of 173 screeningoligonucleotides were selected and they were collectively termed “Round1 oligonucleotides.” These oligonucleotides are listed in Table 1 alongwith their SEQ ID NOs and predicted thermodynamic scores and were latertested in cell culture (Round 1 experiments) for their ability tomodulate CTGF mRNA expression. See Example 6.

Example 2 Antisense Oligonucleotides

The antisense oligonucleotides were synthesized as 20-merphosphorothioate-linked oligodeoxyribonucleotides using standardmethodology (R. Hogrefe. 2010. A short history of oligonucleotidesynthesis. In: TriLink Biotechnologies Nucleic Acid Based Products andServices, pp. 103-108). In some instances, a portion or all of thecytidine residues were replaced, by 5-methyl-2′-deoxycytidine (TriLinkBiotechnologies; San Diego, Calif.). Oligonucleotides were purified byreverse phase or anion exchange high-pressure liquid chromatography(HPLC) with the purity confirmed by polyacrylamide gel elecrtrophoresis,capillary electrophoresis or HPLC. Antisense oligonucleotide identitieswere confirmed by mass spectrometry by the manufacturer (TriLinkBiotechnologies).

In Vitro mRNA Expression Studies

In vitro studies to measure the degree of antisense-mediated inhibitionof human CTGF mRNA were conducted in a systematically iterative manneras outlined below. The studies were performed in three phases termedRound 1, Round 2 and Round 3. Before describing these studies, the celllines, cell culture conditions and transfection parameters are describedin Example 3. The PCR methods for quantitating mRNA expression aredescribed in Example 4. And finally, the steps taken to limit theintroduction of experimental artifacts are described in Example 5.

Round 1 (Example 6) tested the ASOs selected in silico for the highestthermodynamic rankings (Example 1) in a cell-based assay where it wasdiscovered that these antisense oligonucleotides had a mean percentinhibition of CTGF mRNA expression that was similar to that of anegative control ASO. While these results were disappointing, it wasnoted that some ASOs provided a relatively high degree of inhibition ofCTGF mRNA expression (at least 50% reduction) compared tovehicle-treated controls. These were designated as “potent ASOs.” It wasfurther noted that some of the target sequences that are complementaryto potent ASOs appeared to be clustered together suggesting that theremay be regions within the human CTGF mRNA that are hypersensitive toASO-mediated inhibition of mRNA expression, i.e., regions where ASOsknockdown mRNA expression by at least 50%.

Round 2 (Example 8) ASOs were primarily designed based on the Round 1results and were intended to confirm, extend and where possible,identify the limits of these hypersensitive regions of CTGF mRNA. Round3 (Example 9) entailed the testing of ASOs to refine the size andlocations of the hypersensitive regions that were identified, in theearlier two studies.

Example 3 Cell Culture and Transfection

The ability of the synthesized antisense oligonucleotides to modulatethe expression of human CTGF mRNA was tested in human Hs578T breastcarcinoma cells (HTB-126), human A549 lung carcinoma cells (CCL-185) andhuman MG63 osteosarcoma cells (CRL-1427) (American Type CultureCollection (ATCC); Manassas, Va.). The antisense oligonucleotides werealso tested in mouse C2C12 myoblast cells (CRL-1772; ATCC) for theability to modulate the expression of mouse CTGF mRNA.

A549 cells were grown in Kaighn's Modified Ham's F-12 (F-12) Medium(ATCC 30-2004, ATCC) containing 10% fetal bovine serum (FBS; HyClone, S.Logan, Utah). The other cell lines were grown in Dulbecco's ModifiedEagle's Medium (DMEM) (Mediatech, Manassas, Va.) containing 4.5 g/LD-glucose, 584 mg/L L-glutamine, 110 mg/L sodium pyruvate and 10% FBS.Hs578T cells additionally received 5 μg/mL insulin, 5 μg/mL transferrinand 5 ng/mL selenium (Sigma, St. Louis, Mo.).

Cells were initially seeded into 150 cm² tissue culture flasks and oncethey were growing exponentially (66-70% confluent) the cells weredetached with 0.05% trypsin and 0.53 mM EDTA in Hank's Balanced SaltSolution (Mediatech). The cells were then seeded into 96-well tissueculture plates at 1000 cells/well for C2C12 or 2000 cells/well forHs578T, A549 or MG63 in 100 μL of growth media. The cells were culturedin a humidified 37° C. incubator with 5% CO₂ for 24 hrs, after which theculture medium was removed and replaced with 80 μL of fresh growth mediacontaining 10% FBS.

Each well then received 20 μL of antisense oligonucleotide solution. Thesolutions were made by mixing antisense oligonucleotides withLipofectamine™ 2000 (Invitrogen, Carlsbad, Calif.) or DharmaFECT®(Thermo Fisher Scientific, Lafayette, Colo.) transfection reagent inserum-free Opti-MEM® (Invitrogen) according to the transfection reagentmanufacturer's instructions. Final volumes per well of transfectionreagent were of 0.5 μL DharmaFECT® 4(Hs578T), 0.4 μL DharmaFECT® 1 (A549and MG63) or 0.25 μL Lipofectamine™ 2000 (C2C12) and the final antisenseoligonucleotide concentrations were 150 nM.

Cells were cultured for 18 hrs, after which the culture medium wasremoved and the cells washed once with 100 μL of cold (4° C.)phosphate-buffered saline (PBS). After removing the PBS, 50 μL ofTaqMan® Gene Expression Cells-to-CT™ Lysis Solution containing DNase I(Applied Biosystems (ABI) Carlsbad, Calif.) was added to each wellaccording to the manufacturer's instructions. After 5 min of incubationat room temperature, 5 μL of Cells-to-CT™ Stop Solution was added to thelysis reactions and the lysates incubated for another 2 min at roomtemperature. Lysates were then placed on ice for immediate mRNAexpression analysis or stored at −80° C. for later analysis.

Example 4 Real-time Quantitative PGR

cDNA for PGR amplification was generated by reverse transcription (RT)using 10 μL of sample lysate in a 50 μL reaction volume containing IXCells-to-CT™ RT Buffer and IX Cells-to-CT™ RT Enzyme Mix (ABI, P/N4366596). The RT reactions were run for 1 hr at 37° C. The enzyme wasthen inactivated by heating the reaction samples for 5 min at 95° C. RTproducts (cDNA) were diluted 1:5 in nuclease-free water, and real-timequantitative PCR (qPCR) assays were run in triplicate on a 7900HT FastReal-Time PCR System (ABI) using SDS v2.3 and RQ Manager v1.2 softwareaccording to the manufacturer's instructions. Duplex qPCR reactions wereperformed in 10 μL volumes containing 4 μL of diluted cDNA, 5 μL of 2×TaqMan® Gene Expression Master Mix (ABI, P/N 4369016), 0.5 μL ofprimer-limited human β-actin (ACTB) VIC-MGB endogenous control probe(ABI, #4326315E), and 0.5 μL of one of three ABI TaqMan® Gene ExpressionAssays for human CTGF (ABI P/N HS00170014_m1, P/N HS01026927_g1 or P/NHS01026926_g1, which cross the exon4/exon5, exon3/exon4 and exon2/exon3boundaries, respectively). For murine cell lysates, qPCR reactions wererun in separate 10 μL reaction volumes containing 4.5 μL of dilutedcDNA, 5 μL of 2× TaqMan® Gene Expression Master Mix, and either 0.5 μLof mouse β-actin (Actb) Endogenous Control Probe (ABI P/N 4352341E) or0.5 μL of a TaqMan® Gene Expression Assay for mouse Ctgf (ABI P/NMm00515790_g1). Three, technical replicates (qPCR reactions), were runfor each sample (cell lysis reaction).

The murine Ctgf and Actb transcripts were assayed separately and thenActb-normalized Ctgf mRNA expression was calculated from the arithmeticdifference between the median cycle threshold (CT) value for replicateCtgf assays and the median of replicate CT values for endogenous Actbcontrol assays for the same biologic sample (this difference being thedelta CT value). No cross-interference was noted with the simultaneous(duplex) amplification of human CTGF and ACTB transcripts in the sameqPCR well. This allowed for the ACTB-normalized CTGF mRNA expression tobe calculated using the median of the delta CT values for each of thereplicate qPCR wells, where the delta CT value for an individualreaction was calculated as the difference between the CT values for CTGFand ACTB for that specific qPCR reaction. In turn, percent inhibitionfor a specific sample was determined by calculating a ratio ofcalculated relative concentration as compared to actin in a givensample, and then comparing that value to control wells lacking antisenseoligonucleotides.

Results were rejected if the median or well-specific CT values weregreater than 35 cycles for CTGF (i.e., below the limit of CTGF mRNAdetection), or more than 3.32 cycles greater than the median CT valuefor all ACTB amplification curves on that particular qPCR plate for ACTB(i.e., if the ACTB concentration was greater than 10-fold lower than themedian ACTB concentration for all assays run on the same qPCR plate).All duplex assays provided CTGF, ACTB and ACTB-normalized CTGF valuesthat were closely correlated with those obtained from assays where eachtarget was quantitated separately (r=0.95-0.99, p<1e-6), thus indicatinga lack of interference between the combined target (CTGF) and endogenouscontrol (ACTB) assays. “Minus-RT” controls containing all RT reactioncomponents except the 20× RT Enzyme Mix and “minus-template” controlscontaining all qPCR reagents but no cell lysate were consistentlynegative for any amplification signal due to genomic DNA or PCR productcarry-over contamination.

Example 5 Avoidance of Systematic Technical Artifacts

Multiple precautions were taken to avoid the introduction of systematicstudy artifacts during the antisense oligonucleotide assays. To avoidsystematic PCR plate position effects all antisense oligonucleotides andcontrols were delivered to randomly assigned distinct tissue culturewells in each experiment so that no antisense oligonucleotide was everdelivered to the same well across independent experiments. ASOs elicitedsimilar levels of inhibition of CTGF mRNA expression (knockdown) acrossall experiments (r=0.48-0.86, p<1e-6 for any two Hs578T experiments),indicating that inhibition was not related to PCR plate location.

In addition, PBS vehicle control was delivered to different wellpositions in each experiment, and no systematic differed in baselineCTGF mRNA expression was observed within (mean CV=13%) of between (ANOVAp=0.78) any of the screening experiments. Likewise, there was excellentagreement between plates within each individual experiment (r>0.92) asdetermined through the use of “bridging” controls, i.e., the samecontrol ASOs were delivered to two independent cell culture plates inthe sane individual experiment. Similarly, there was excellent agreementbetween Rounds (r=0.86), as indicated by the use of 29 Round 1 and 25Round 2 inter Round “bridging” ASOs in Round 3 experiments.

A systematic analysis of antisense data obtained from wells locatedalong the periphery of a tissue culture plate versus those that camefrom internal wells also failed to reveal any difference relating toplate position, again consistent with the observation that the variousantisense oligonucleotides exerted similar antisense effects from oneexperiment to another regardless of which tissue culture well they weredelivered to. Thus, no systematic technical artifacts were detected.

It has been reported that ASO carryover can sometimes systematicallyinterfere with PCR detection of transcript levels (Frier S M and Watt AT, Basic Principles of Antisense Drug Discovery. In: Antisense DrugTechnology: Principles, Strategies, and Applications 2nd Ed., S TCrooke, ed., 2008, CRC Press New York, pp 117-141), particularly whenthe ASO is designed to target sequences located within the PGR amplicon.To investigate this potential artifact, multiple CTGF qPCR assaysdirected towards different regions of the CTGF transcript wereevaluated. Excellent agreement between independent CTGF qPCR assaysHS00170014_m1 and HS01026927_g1 was observed (r=0.78-0.86 for all Round1 screening ASOs and experiments). However, the latter assay didindicate moderate but consistently greater magnitude of CTGF targetedmRNA knockdown that the former assay for selected ASOs that targetedCTGF mRNA bases 721-767 surrounding the exon3/exon4 splice site (i.e.,for sequences within the HS01026927_g1 amplicon). Otherwise, these twoassays were in close agreement over all other CTGF mRNA regions,including the region from bases 931-987 that overlapped theHS00170014_m1 amplicon and including all regions that demonstratedhypersensitivity to ASO-mediated inhibition as disclosed in subsequentExamples (i.e., SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ IDNO: 321, SEQ ID NO: 322; SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325,SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ IDNO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334,SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ IDNO: 339, SEQ ID NO: 340, Table 3).

For the relatively small number of Round 1 oligonucleotides (11) thatexhibited poor-to-modest agreement between the HS00170014_m1 andHS01026927_g1 assays, a third TaqMan® qPCR assay (HS01026926_g1) thatcrosses the exon2/exon3 boundary was tested and demonstrated closeagreement with the HS01026927_g1 assay. All three assays were in closeagreement with each other for oligonucleotides that already showedreasonable agreement between the HS00170014_m1 and HS01026927_g1 assays,including within all hypersensitive regions (vide infra).

To minimize outlier effects due to occasional differences in assaybehavior, the level of β-actin-normalized CTGF mRNA inhibition providedby a given ASO in a particular Round 1 experiment was calculated as themedian of the percent inhibition values for all independent qPCR assaysrun for that particular ASO. Given the overall uniformity of results forthe three TaqMan® assays used in the Round 1 experiments, CTGF mRNAexpression levels in subsequent Round 2 and Round 3 studies weredetermined using the HS00170014_m1 assay. The overall percent CTGF mRNAinhibition for a given antisense oligonucleotide was in turn, calculatedas the median of the percent inhibition values obtained in three to tenindependent Round 1, Round 2 and/or Round 3 experiments, in which thatparticular antisense oligonucleotide was analyzed.

Example 6 Round 1 Experiments

The 173 screening antisense oligonucleotides (20-mers) selected insilico, as described in Example 1, were synthesized as described inExample 2. The thermodynamic scores for the Round 1 ASOs ranged from−1.45 to 7.63 (median=1.65, mean±SD=1.75±1.62). These antisenseoligonucleotides were predicted to produce the greatest degree of CTGFmRNA inhibition per set of antisense oligonucleotides to individualscreening windows. The antisense oligonucleotides were tested in Hs578Tcells for their ability to modulate expression of CTGF mRNA. Data foreach antisense oligonucleotide were obtained in three independentexperiments, collectively termed “Round 1 experiments” or “Round 1studies.” The calculated percent inhibition from the combinedexperiments for each oligonucleotides listed in Table 1.

TABLE 1 Round 1 Antisense Oligonucleotides CTGF mRNA Inhibition 5′ 3′Thermo- Motif- % Target Target dynamic based Inhibition Site SiteAntisense Sequence Score Score in Round 1 SEQ ID NO 5 24ggggaagagttgttgtgtga 2.74 −0.2 18.9 SEQ ID NO: 2 30 49gtcgcactggctgtctcctc 4.00 −0.2 21.1 SEQ ID NO: 3 44 63agctggagggtggagtcgca 3.51 0 37.4 SEQ ID NO: 4 55 74 ggctgccgtcgagctggagg2.24 0 31.9 SEQ ID NO: 5 75 94 tcggggctgtcggccggggc 1.75 −0.5 6.6SEQ ID NO: 6 85 104 ggctgtcgtctcggggctgt 4.19 −0.2 17.0 SEQ ID NO: 7 199218 GGCGGCGGTCATggttggca 3.01 0 −35.8 SEQ ID NO: 8 210 229GGGCCCATACTGGCGGCGGT 2.02 −0.2 −0.5 SEQ ID NO: 9 217 236GCGGACGGGGCCCATACTGG 1.92 −0.4 21.2 SEQ ID NO: 10 222 241GCGACGCGGACGGGGCCCAT 1.86 −0.2 42.5 SEQ ID NO: 11 239 258CGAGGAGGACCACGAAGGCG 1.07 0.3 14.9 SEQ ID NO: 12 246 265CAGAGGGCGAGGAGGACCAC 2.56 0.3 12.8 SEQ ID NO: 13 253 272CCGGCTGCAGAGGGCGAGGA 0.95 −0.1 27.8 SEQ ID NO: 14 264 283CCGACGGCCGGCCGGCTGCA −1.14 −0.2 14.8 SEQ ID NO: 15 280 299CCCGCTGCAGTTCTGGCCGA 3.03 0 −1.3 SEQ ID NO: 16 284 303ACGGCCCGCTGCAGTTCTGG 2.49 0 5.8 SEQ ID NO: 17 314 333AGCGCGGCGCCGGCTCGTCC −0.02 −0.1 −33.4 SEQ ID NO: 18 338 357GCACGAGGCTCACGCCCGCC 1.27 0 30.2 SEQ ID NO: 19 350 369CGCAGCCGTCCAGCACGAGG 3.07 0 3.8 SEQ ID NO: 20 354 373CAGCCGCAGCCGTCCAGCAC 4.88 0 17.0 SEQ ID NO: 21 357 376CAGCAGCCGCAGCCGTCCAG 4.15 0 −2.5 SEQ ID NO: 22 358 377GCAGCAGCCGCAGCCGTCCA 4.29 0 6.4 SEQ ID NO: 23 370 389GGCGCAGACGCGGCAGCAGC −0.01 0 38.2 SEQ ID NO: 24 381 400CCCAGCTGCTTGGCGCAGAC 0.78 0 48.1 SEQ ID NO: 25 387 406AGCTCGCCCAGCTGCTTGGC 0.97 0 31.1 SEQ ID NO: 26 397 416CTCGGTGCACAGCTCGCCCA 2.76 0 42.5 SEQ ID NO: 27 409 428GCATGGGTCGCGCTCGGTGC 3.15 0 −134.3 SEQ ID NO: 28 415 434CGGGTCGCATGGGTCGCGCT 2.59 0 45.8 SEQ ID NO: 29 430 449GAAGAGGCCCTTGTGCGGGT 1.95 0 31.0 SEQ ID NO: 30 436 455GTCACAGAAGAGGCCCTTGT 1.32 0 34.9 SEQ ID NO: 31 447 466GGGGAGCCGAAGTCACAGAA 2.24 −0.2 16.4 SEQ ID NO: 32 460 479CTTGCGGTTGGCCGGGGAGC 1.57 −0.3 −64.2 SEQ ID NO: 33 467 486CGCCGATCTTGCGGTTGGCC 1.65 0 9.8 SEQ ID NO: 34 475 494GGTGCACACGCCGATCTTGC 0.80 0 6.4 SEQ ID NO: 35 486 505CCATCTTTGGCGGTGCACAC 0.81 0 25.7 SEQ ID NO: 36 496 515GCAGGGAGCACCATCTTTGG 1.65 0 20.8 SEQ ID NO: 37 507 526CCACCGAAGATGCAGGGAGC 2.37 0.3 35.3 SEQ ID NO: 38 514 533CACCGTACCACCGAAGATGC 3.11 0.3 23.9 SEQ ID NO: 39 528 547TCTCCGCTGCGGTACACCGT 0.05 0 7.4 SEQ ID NO: 40 537 556TGGAAGGACTCTCCGCTGCG 0.88 0.3 36.2 SEQ ID NO: 41 554 573GGTACTTGCAGCTGCTCTG −0.77 0.1 38.1 SEQ ID NO: 42 567 586AGGCACGTGCACTGGTACTT −0.63 −0.2 61.9 SEQ ID NO: 43 580 599CACCGCCCCGTCCAGGCACG 4.09 0 16.3 SEQ ID NO: 44 584 603AGCCCACCGCCCCGTCCAGG 7.63 0.3 16.2 SEQ ID NO: 45 589 608CATGCAGCCCACCGCCCCGT 6.20 0.3 23.9 SEQ ID NO: 46 600 619CTGCACAGGGGCATGCAGCC −0.19 −0.2 19.7 SEQ ID NO: 47 608 627CGTCCATGCTGCACAGGGGC 1.14 −0.2 0.2 SEQ ID NO: 48 613 632ACGAACGTCCATGCTGCACA 1.29 0 56.6 SEQ ID NO: 49 629 648AGTCAGGGCTGGGCAGACGA 2.95 0 33.7 SEQ ID NO: 50 639 658GGGAAGGGGCAGTCAGGGCT 4.31 −0.2 13.4 SEQ ID NO: 51 646 665CCTCCTCGGGAAGGGGCAGT 2.39 −0.2 3.2 SEQ ID NO: 52 657 676GGCAGCTTGACCCTCCTCGG 4.57 0 29.0 SEQ ID NO: 53 661 680CCCGGGCAGCTTGACCCTCC 2.82 −0.1 44.7 SEQ ID NO: 54 677 696ACTCCTCGCAGCATTTCCCG 3.92 0.5 29.6 SEQ ID NO: 55 684 703CACACCCACTCCTCGCAGCA 4.36 0.5 51.1 SEQ ID NO: 56 688 707GTCACACACCCACTCCTCGC 4.85 0.5 17.8 SEQ ID NO: 57 692 711GCTCGTCACACACCCACTCC 4.70 0.5 44.3 SEQ ID NO: 58 710 729CCACGGTTTGGTCCTTGGGC −1.42 0.3 34.5 SEQ ID NO: 59 721 740GGCAGGCCCAACCACCGTTT 3.08 0.3 65.8 SEQ ID NO: 60 737 756GTCGGTAAGCCGCGAGGGCA −0.70 −0.2 28.5 SEQ ID NO: 61 747 766GTGTCTTCCAGTCGGTAAGC 2.23 −0.2 68.9 SEQ ID NO: 62 754 773GCCAAACGTGTCTTCCAGTC 2.37 0.1 31.4 SEQ ID NO: 63 762 781GGGTCTGGGCCAAACGTGTC 1.19 0.1 12.5 SEQ ID NO: 64 772 791AATCATAGTTGGGTCTGGGC 2.08 0 33.2 SEQ ID NO: 65 791 810GGACCAGGCAGTTGGCTCTA 2.08 0.1 74.8 SEQ ID NO: 66 809 828CGCTCCACTCTGTGGTCTGG 0.69 0.6 27.2 SEQ ID NO: 67 817 836GGAACAGGCGCTCCACTCTG 1.47 0.6 47.7 SEQ ID NO: 68 836 855TGCCCATCCCACAGGTCTTG 4.44 0.6 9.1 SEQ ID NO: 69 841 860GGAGATGCCCATCCCACAGG 2.28 0.6 36.8 SEQ ID NO: 70 859 878GTCATTGGTAACCCGGGTGG 0.06 −0.3 78.5 SEQ ID NO: 71 865 884GGCGTTGTCATTGGTAACCC 2.49 −0.2 44.0 SEQ ID NO: 72 870 889CAGGAGGCGTTGTCATTGGT 2.95 0 29.4 SEQ ID NO: 73 889 908GCTCTGCTTCTCTAGCCTGC 3.00 0.2 13.8 SEQ ID NO: 74 893 912GGCGGCTCTGCTTCTCTAGC 2.06 0.2 51.6 SEQ ID NO: 75 904 923GACCATGCACAGGCGGCTCT 1.87 0.1 50.4 SEQ ID NO: 76 912 931CAAGGCCTGACCATGCACAG 0.91 0 75.6 SEQ ID NO: 77 931 950CTCTTCCAGGTCAGCTTCGC 3.50 0.1 25.0 SEQ ID NO: 78 935 954TGTTCTCTTCCAGGTCAGCT 3.02 0.1 51.6 SEQ ID NO: 79 946 965GCCCTTCTTAATGTTCTCTT 2.91 −0.1 26.9 SEQ ID NO: 80 960 979CGGATGCACTTTTTGCCCTT 1.76 0 74.7 SEQ ID NO: 81 967 986GGGAGTACGGATGCACTTTT 1.49 0 75.2 SEQ ID NO: 82 981 1000GGCTTGGAGATTTTGGGAGT 2.49 0 58.2 SEQ ID NO: 83 989 1008ACTTGATAGGCTTGGAGATT 0.82 0 64.1 SEQ ID NO: 84 995 1014GCTCAAACTTGATAGGCTTG 0.22 −0.1 38.7 SEQ ID NO: 85 1003 1022GCCAGAAAGCTCAAACTTGA 0.59 0 38.1 SEQ ID NO: 86 1006 1025GCAGCCAGAAAGCTCAAACT 1.14 0 62.4 SEQ ID NO: 87 1019 1038TCTTCATGCTGGTGCAGCCA 0.31 0.2 72.6 SEQ ID NO: 88 1029 1048GCTCGGTATGTCTTCATGCT 1.02 0 47.0 SEQ ID NO: 89 1036 1055GAATTTAGCTCGGTATGTCT 0.79 0 65.1 SEQ ID NO: 90 1043 1062CTCCACAGAATTTAGCTCGG 1.39 0.3 65.9 SEQ ID NO: 91 1057 1076GCCGTCGGTACATACTCCAC 1.98 0.5 66.2 SEQ ID NO: 92 1068 1087GTGCAGCATCGGCCGTCGGT 1.76 0 74.4 SEQ ID NO: 93 1071 1090GGGGTGCAGCATCGGCCGTC 1.63 −0.2 49.3 SEQ ID NO: 94 1074 1093TGGGGGGTGCAGCATCGGCC 3.13 −0.2 37.1 SEQ ID NO: 95 1081 1100GGTTCTGTGGGGGGTGCAGC 3.43 −0.2 22.3 SEQ ID NO: 96 1088 1107GGGTGGTGGTTCTGTGGGGG 5.27 −0.2 −11.4 SEQ ID NO: 97 1093 1112CGGCAGGGTGGTGGTTCTGT 3.57 0 42.0 SEQ ID NO: 98 1109 1128GGCACTTGAACTCCACCGGC 2.67 0.4 80.4 SEQ ID NO: 99 1122 1141ACCTCGCCGTCAGGGCACTT 0.64 0 68.2 SEQ ID NO: 100 1132 1151CTTCTTCATGACCTCGCCGT 2.68 0 59.8 SEQ ID NO: 101 1142 1161ACATCATGTTCTTCTTCATG −0.66 0 −4.2 SEQ ID NO: 102 1159 1178GGCACAGGTCTTGATGAACA −1.18 0 64.4 SEQ ID NO: 103 1168 1187GTAATGGCAGGCACAGGTCT 1.84 −0.2 66.4 SEQ ID NO: 104 1179 1198CCGGGACAGTTGTAATGGCA 1.87 −0.3 77.9 SEQ ID NO: 105 1193 1212AGATGTCATTGTCTCCGGGA 1.57 −0.1 69.4 SEQ ID NO: 106 1205 1224ACAGCGATTCAAAGATGTCA 0.59 −0.1 31.9 SEQ ID NO: 107 1211 1230TGTAGTACAGCGATTCAAAG −0.95 −0.1 58.5 SEQ ID NO: 108 1228 1247GTCTCCGTACATCTTCCTGT 2.91 0 27.3 SEQ ID NO: 109 1233 1252GCCATGTCTCCGTACATCTT 2.69 0.2 40.0 SEQ ID NO: 110 1240 1259gctTCATGCCATGTCTCCGT 2.79 0.2 68.1 SEQ ID NO: 111 1249 1268cactctctggctTCATGCCA −0.79 0.5 36.3 SEQ ID NO: 112 1255 1274gtctctcactctctggctTC 3.34 0.4 46.7 SEQ ID NO: 113 1261 1280gttaatgtctctcactctct 1.40 0.2 42.5 SEQ ID NO: 114 1286 1305atcagttcaagttccagtct 2.49 0 33.7 SEQ ID NO: 115 1305 1324acggaaaaatgagatgtgaa −1.03 −0.1 17.3 SEQ ID NO: 116 1320 1339actgaaatcatttttacgga −0.85 −0.3 −0.7 SEQ ID NO: 117 1329 1348acttgtgctactgaaatcat 0.42 −0.3 58.3 SEQ ID NO: 118 1357 1376cccccagttagaaaaacaga 1.26 −0.1 25.1 SEQ ID NO: 119 1366 1385gaatcttttcccccagttag 3.10 0.3 47.1 SEQ ID NO: 120 1384 1403atgttttgaattgggtggga 1.41 0 5.5 SEQ ID NO: 121 1405 1424ctatttgtttgacatggcac −0.70 0 43.0 SEQ ID NO: 122 1417 1436ggggttgatagactatttgt 0.41 −0.2 9.9 SEQ ID NO: 123 1425 1444cagtgtctggggttgataga 2.10 −0.2 43.3 SEQ ID NO: 124 1437 1456cattcttcaaaccagtgtct 1.31 −0.1 14.0 SEQ ID NO: 125 1455 1474tccactgtcaagtcttaaca 0.90 −0.1 23.7 SEQ ID NO: 126 1460 1479gtagttccactgtcaagtct 2.33 0.1 49.7 SEQ ID NO: 127 1483 1502acattctggtgctgtgtact 1.93 0 48.4 SEQ ID NO: 128 1498 1517gccacaccttaatatacatt 1.48 0.3 48.2 SEQ ID NO: 129 1514 1533tcccactgctcctaaagcca 2.95 0.3 27.9 SEQ ID NO: 130 1520 1539gtaccctcccactgctccta 5.23 0.4 64.5 SEQ ID NO: 131 1527 1546tctgctggtaccctcccact 3.49 0.6 64.4 SEQ ID NO: 132 1531 1550cctttctgctggtaccctcc 3.21 0 59.4 SEQ ID NO: 133 1549 1568gctatctgatgatactaacc −0.20 −0.2 46.0 SEQ ID NO: 134 1574 1593agcaggcatattactcgtat 1.21 0.2 52.0 SEQ ID NO: 135 1585 1604acacttcaaatagcaggcat 0.73 −0.1 41.0 SEQ ID NO: 136 1597 1616tccttctcaattacacttca 1.48 0 14.9 SEQ ID NO: 137 1620 1639ggtcagtgagcacgctaaaa 0.42 −0.3 40.3 SEQ ID NO: 138 1633 1652ggggctacaggcaggtcagt 3.32 −0.2 72.2 SEQ ID NO: 139 1647 1666tcctagctgtcactggggct 2.88 −0.4 48.3 SEQ ID NO: 140 1653 1672tgcacatcctagctgtcact 2.47 0 60.4 SEQ ID NO: 141 1670 1689tcttgatggctggagaatgc 1.89 0 49.3 SEQ ID NO: 142 1681 1700ttgactcagtctcttgatgg 0.07 0.3 10.1 SEQ ID NO: 143 1712 1731ctgagtctgctgttctgact 0.98 0 30.9 SEQ ID NO: 144 1740 1759cagtgtcattcgaatcagaa −1.15 0 37.4 SEQ ID NO: 145 1752 1771ccgattcctgaacagtgtca 1.44 0 59.5 SEQ ID NO: 146 1763 1782tcgacaggattccgattcct 0.96 0 62.8 SEQ ID NO: 147 1783 1802ccacaagctgtccagtctaa 1.85 0.1 68.4 SEQ ID NO: 148 1785 1804tgccacaagctgtccagtct 2.76 0.5 74.8 SEQ ID NO: 149 1789 1808cacttgccacaagctgtcca 1.22 0.5 70.4 SEQ ID NO: 150 1803 1822gttacaggcaaattcacttg 0.62 −0.1 20.3 SEQ ID NO: 151 1892 1911attaacttagtataactgtac −1.45 −0.6 −3.3 SEQ ID NO: 152 1909 1928ggcacaaacaagtttaaatt −1.08 −0.4 26.9 SEQ ID NO: 153 1962 1981cagaaattgaggctaacatt −0.77 −0.3 16.6 SEQ ID NO: 154 1980 1999cattctacctatggtgttca 1.13 0 41.8 SEQ ID NO: 155 1986 2005gctttacattctacctatgg 1.64 0 23.7 SEQ ID NO: 156 1998 2017acgatcagacaagctttaca −0.32 0 34.5 SEQ ID NO: 157 2018 2037gtatccatttcatgctttga 1.17 0 33.6 SEQ ID NO: 158 2026 2045ccatataagtatccatttca 0.99 −0.2 22.0 SEQ ID NO: 159 2052 2071actgtcattctatctgagca 1.43 −0.2 33.3 SEQ ID NO: 160 2056 2075acggactgtcattctatctg 0.18 −0.2 18.8 SEQ ID NO: 161 2086 2105atgcctcccctttgcaaaca 0.12 0.2 26.5 SEQ ID NO: 162 2091 2110cactgatgcctcccctttgc 4.85 0.1 53.8 SEQ ID NO: 163 2115 2134acctagaaatcagcctgcca 2.54 0.1 36.0 SEQ ID NO: 164 2131 2150ggctaccacatttcctacct 3.62 0.3 32.6 SEQ ID NO: 165 2153 2172gccatttgttcattaaaagt 0.53 −0.1 11.1 SEQ ID NO: 166 2175 2194agtcactcagtttttaataa −0.20 −0.2 19.4 SEQ ID NO: 167 2184 2203gctatatagagtcactcagt −0.35 0.2 22.1 SEQ ID NO: 168 2208 2227gcttccaggtgaaaaaactg 0.54 −0.4 22.8 SEQ ID NO: 169 2215 2234aacaaatgcttccaggtgaa 0.74 −0.1 55.8 SEQ ID NO: 170 2221 2240agtagaaacaaatgcttcca 0.07 −0.2 5.6 SEQ ID NO: 171 2243 2262gtccgaaaaacagtcatatc 0.47 −0.1 43.4 SEQ ID NO: 172 2258 2277ctcaacaaataaactgtccg −0.04 −0.6 39.2 SEQ ID NO: 173 2311 2330atacactttattttcaacta 0.10 0 31.0 SEQ ID NO: 174 Table 1. Thermodynamicand motif-base scores for 173 Round 1 screening oligonucleotides andtheir median % inhibition of CTGF mRNA expression vs. vehicle-treatedcontrols from three combined experiments. To highlight the lack of anassociation between the predicted and empirical outcomes, thermodynamicscores ≧4 (i.e., those that should have forecast the greatest level ofantisense activity) are underlined and bolded, as are inhibition values≧50%. The 5′ and 3′ target sites denote the 5′-most and 3′-mostcomplementary (sense) bases of the spliced human CTGF mRNA sequence, SEQID NO: 1, for the 20-nucleotide-long-ASOs. An “m” suffix denotes anoligonucleotide that crosses an exon-exon boundary and that is thus onlycomplementary to the spliced mRNA. All other ASOs are complementary toboth the spliced mRNA and non-spliced pre-mRNA.

Surprisingly, despite the careful selection based on calculatedthermodynamic properties, the Round 1 antisense oligonucleotidesproduced mean and median percent inhibitions of human CTGF mRNAexpression of 34.1%, and 34.5%, respectively, that were equivalent tothat produced by a negative control antisense oligonucleotide to thegene phosphatase and tensin homolog (PTEN). A shift in the mean andmedian percent inhibition above that produced by the negative control,i.e., above 34%, was expected for this group as they were selected outof 2325 ASOs based on favorable thermodynamic characteristics thatcorrelate with hybridization. These results suggested that the degree ofinhibition of CTGF mRNA expression produced by the selected ASOs werenon-specific and that the thermodynamic-based selection algorithm couldnot identify antisense oligonucleotides that had a high degree ofinhibitory activity.

The failure of the calculated thermodynamic scores to predict antisenseoligonucleotide sequences with high inhibitory activity is exemplifiedby the absence of a correlation between the predicted scores andobserved outcomes (r=−0.04). For example, 17 of the Round 1 ASOs had acalculated thermodynamic score of at least 4 and were therefore over 1standard deviation above the mean for the entire set of Round 1 ASOs.These ASOs were thus predicted to possess the strongest inhibitoryactivity (Table 1). Surprisingly, only three of these 17 ASOs (SEQ IDNO: 56, SEQ ID NO: 131, and SEQ ID NO: 163) inhibited CTGFmRNA-expression by at least 5.0% in the Round 1 studies. Conversely,another 14 Round 1 ASOs with otherwise exemplary thermodynamic scoresprovided only 16±4 percent mRNA inhibition, (mean±SE). Thus, in terms offorecasting which ASOs would provide at least 50% inhibition of CTGFmRNA expression, a thermodynamic cut-off score of 4 had a positivepredictive value of only 18% and a false discovery rate of 82%. Indeed,the area under the receiver operating characteristic (ROC) curvedescribing the ability of the thermodynamic score to identify ASOs thatprovide at least 50% CTGF mRNA knockdown was 0.52, thus indicating thatthe thermodynamic score had no predictive utility (p=0.69).

A further observation that exemplifies the lack of predictive power ofthe thermodynamic score to identify ASOs with high inhibitory activityis that none of the 22 Round 1 ASOs exhibiting the highest inhibitoryactivity (at least 65% mRNA inhibition) were predicted to have such highactivity. Instead, of the thermodynamic scores for these ASOs ranked26th to 147th among the 173 Round 1 ASOs (median rank=81.5), fallinginto no clear pattern. Likewise, adjusting the ROC curve calculations toexamine the ability of thermodynamic score to identify ASOs that provideat least 65% CTGF mRNA knockdown also revealed that the thermodynamicscore was not predictive (p=0.85).

Given the failure of the thermodynamic algorithm to predict forantisense oligonucleotide activity, other parameters were evaluated toexamine whether they correlated with the observed antisense activity ofindividual ASOs. No correlations were found between the observed levelof CTGF mRNA inhibition and the calculated intra-oligonucleotide,inter-oligonucleotide or oligonucleotide-to-target free energy values(r=0.07-0.14). Additionally, a correlation coefficient-weighted scorebased on the presence of sequence motifs previously associated withantisense activity (Matveeva et al. Nucleic Acids Res 2000 28:2862-5)was compared with the observed percent inhibition data. This motif-basedscore was calculated using the following equation: motif-basedscore=(0.3×the number of CCAC motifs+0.3×the number of TCCCmotifs+0.2×the number of ACTC motifs+0.2×the number of GCCAmotifs+0.1×the number of CTCT motifs)−(0.2×the number of GGGGmotifs−0.2×the number of ACTG motifs−0.2×the number of TAAmotifs−0.1×the number of CCGG motifs−0.1×the number of AAA motifs) in agiven ASO. Like the thermodynamic score and component free energyvalues, this motif-based score was not associated with the level ofantisense activity observed in the Round 1 studies (Spearman r=0.22),Table 1. The motif-based score was also a rather weak predictor of ASOsthat caused at least 50% CTGF mRNA knockdown, since the area under theROC curve describing its ability to do so (0.60) was not substantiallydifferent from the area under the diagonal line of no discriminationLikewise a TCCC sequence motif was only present in 3 (7%) of the 44 ASOsthat provided at least 50% inhibition, even though it had beenidentified in 48% of the most potent ASOs reported at one point in theliterature and 59% of ASOs containing this motif were active in aprospective study aimed at validating its predictive utility (Tu et al.J Biol Chem 1998 273:25125-31).

Example 7 Non-random Distribution of Potent Sequences with Respect toLocation on the CTGF mRNA

Notably, rather than exhibiting a normal (Gaussian) distribution about asingle mean, the Round 1 data appeared to resemble a bi-phasicdistribution, as illustrated by a histogram of the inhibitory activityof Round 1 ASOs (FIG. 2). Here, a clear shoulder can be seen to theright side of an otherwise broad bell-shaped distribution that suggestedthe presence of a sub-population of ASOs with strong inhibitor activity.This suggestion was supported by a data inflection point atapproximately 50% inhibition shown on a plot of percent inhibitionversus rank for the Round 1 ASOs (FIG. 3). Based on this graph,antisense oligonucleotides that produced at least 50% inhibition ofhuman CTGF mRNA expression were defined as “potent” antisenseoligonucleotides.

Furthermore, as can be seen in Table 1, the potent ASOs (i.e., thosethat caused at least 50% CTGF mRNA inhibition in Round 1) were notrandomly distributed in terms of the locations of their complementarytarget sequences along the CTGF mRNA. Instead, from these Round 1results, several clusters of sequences that are complementary to potentASOs appear to be present, suggestive of contiguous local regions (i.e.,beyond 20 nucleotides in length) of the human CTGF mRNA that arehypersensitive to antisense inhibition of mRNA expression. Thesehypersensitive regions may be structurally more accessible to antisenseoligonucleotides. Hypersensitive regions suggested by Round 1 ASO dataincluded regions corresponding to nucleotides 893-931, 960-1008,1006-1038, 1036-1087, 1109-1151, 1159-1212, 1520-1550 and 1752-1808 ofthe human CTGF mRNA sequence.

Example 8 Round 2 Experiments

In response to the suggestion of hypersensitive regions in Example 7,further experiments were performed to confirm, extend and wherepossible, identify the limits of these suspected hypersensitive regions.To this end, 77 additional “Round 2” oligonucleotides (Table 2) weredesigned that mapped to the regions that surround and/or overlap mRNAsequences that were complementary to 30 “seed” antisenseoligonucleotides from Round 1 experiments (Table 1). These seedoligonucleotides inhibited CTGF mRNA expression by at least 60% in Round1 experiments.

The number of Round 2 oligonucleotides tested for each seedoligonucleotide depended, in part, on the inhibitory activity of theRound 1 oligonucleotides that are adjacent to a specific seedoligonucleotide. For example, FGTC-1240-PS20 (SEQ ID NO: 111) elicited68.1% CTGF mRNA knockdown in Round 1 experiments and is surrounded byfour other Round 1 ASOs that overlap at least five of the same targetbases as FGTC-1240-PS20. These adjacent ASOs, however, provided only27.3% to 46.7% knockdown (mean=37.6%). Therefore, only one flankingRound 2 ASO was synthesized on each side of FGTC-1240-PS20, SEQ ID NO:111, as the size of a potential hypersensitive region appeared to belimited. On the other hand, FGTC-1122-PS20 (SEQ ID NO: 100) elicitedessentially the same degree of mRNA inhibition as FGTC-1240-PS20, SEQ IDNO: 111, (68.2%), but was surrounded by two Round 1 ASOs with at leastfive overlapping target bases that provided 59.8% and 80.4% CTGF mRNAknockdown. Because this local region appeared unusually enriched, withpotent ASO sequences with the potential to represent an extendedhypersensitive region, six ASOs were selected for Round 2 testing toprobe the region surrounding FGTC-1122-PS20, SEQ ID NO: 100.

Each of the Round 2 ASOs was tested in four independent Round 2experiments conducted in Hs578T cells, the results of which aresummarized in Table 2. Notably, a significantly higher proportion ofRound 2 than Round 1 ASOs elicited ≧50% inhibition of CTGF mRNAexpression (p=3e-10). Thus, the Round 2 ASOs, which were selectedbecause they surrounded the best Round 1 ASOs, were highly enriched forpotent ASOs as compared to the starting set of Round 1 screening ASOs.This shift in distributions is shown by comparing FIG. 2 with FIG. 4.The Round 2 data further support the existence of hypersensitivityregions and demonstrates that they are larger than predicted based juston Round 1 data.

TABLE 2 Round 1-3 Antisens Oligonucleotides CTGF mRNA Inhibition 5′ 3′Target Target % Regional Round Site Site Antisense Sequence InhibitionStatus SEQ ID NO 1 5 24 ggggaagagttgttgtgtga 18.9 SEQ ID NO: 2 1 30 49gtcgcactggctgtctcctc 21.1 SEQ ID NO: 3 1 44 63 agctggagggtggagtcgca 37.4SEQ ID NO: 4 1 55 74 ggctgccgtcgagctggagg 31.9 SEQ ID NO: 5 1 75 94tcggggctgtcggccggggc 6.6 SEQ ID NO: 6 1 85 104 ggctgtcgtctcggggctgt 17.0SEQ ID NO: 7 1 199 218 GGCGGCGGTCATggttggca −18.6 SEQ ID NO: 8 1 210 229GGGCCCATACTGGCGGCGGT −0.5 SEQ ID NO: 9 1 217 236 GCGGACGGGGCCCATACTGG21.2 SEQ ID NO: 10 1 222 241 GCGACGCGGACGGGGCCCAT 42.5 SEQ ID NO: 11 1239 258 CGAGGAGGACCACGAAGGCG 14.9 SEQ ID NO: 12 1 246 265CAGAGGGCGAGGAGGACCAC 12.8 SEQ ID NO: 13 1 253 272 CCGGCTGCAGAGGGCGAGGA26.3 SEQ ID NO: 14 1 264 283 CCGACGGCCGGCCGGCTGCA 14.8 SEQ ID NO: 15 1280 299 CCCGCTGCAGTTCTGGCCGA −1.3 SEQ ID NO: 16 1 284 303ACGGCCCGCTGCAGTTCTGG 5.8 SEQ ID NO: 17 1 314 333 AGCGCGGCGCCGGCTCGTCC−16.4 SEQ ID NO: 18 1 338 357 GCACGAGGCTCACGCCCGCC 30.2 SEQ ID NO: 19 1350 369 CGCAGCCGTCCAGCACGAGG 3.8 SEQ ID NO: 20 1 354 373CAGCCGCAGCCGTCCAGCAC 17.0 SEQ ID NO: 21 1 357 376 CAGCAGCCGCAGCCGTCCAG−2.5 SEQ ID NO: 22 1 358 377 GCAGCAGCCGCAGCCGTCCA 6.4 SEQ ID NO: 23 1370 389 GGCGCAGACGCGGCAGCAGC 38.2 SEQ ID NO: 24 1 381 400CCCAGCTGCTTGGCGCAGAC 48.1 SEQ ID NO: 25 1 387 406 AGCTCGCCCAGCTGCTTGGC31.1 SEQ ID NO: 26 1 397 416 CTCGGTGCACAGCTCGCCCA 42.5 SEQ ID NO: 27 1409 428 GCATGGGTCGCGCTCGGTGC −72.2 SEQ ID NO: 28 1 415 434CGGGTCGCATGGGTCGCGCT 45.8 SEQ ID NO: 29 1 430 449 GAAGAGGCCCTTGTGCGGGT31.0 SEQ ID NO: 30 1 436 455 GTCACAGAAGAGGCCCTTGT 34.9 SEQ ID NO: 31 1447 466 GGGGAGCCGAAGTCACAGAA 16.4 SEQ ID NO: 32 1 460 479CTTGCGGTTGGCCGGGGAGC −57.3 SEQ ID NO: 33 1 467 486 CGCCGATCTTGCGGTTGGCC9.8 SEQ ID NO: 34 1 475 494 GGTGCACACGCCGATCTTGC 6.4 SEQ ID NO: 35 1 486505 CCATCTTTGGCGGTGCACAC 25.0 SEQ ID NO: 36 1 496 515GCAGGGAGCACCATCTTTGG 20.8 SEQ ID NO: 37 1 507 526 CCACCGAAGATGCAGGGAGC35.3 SEQ ID NO: 38 1 514 533 CACCGTACCACCGAAGATGC 23.9 SEQ ID NO: 39 1528 547 TCTCCGCTGCGGTACACCGT 7.4 SEQ ID NO: 40 1 537 556TGGAAGGACTCTCCGCTGCG 36.2 SEQ ID NO: 41 1 554 573 GGTACTTGCAGCTGCTCTGG38.1 SEQ ID NO: 42 2 565 584 GCACGTGCACTGGTACTTGC 28.4 SEQ ID NO: 175 1567 586 AGGCACGTGCACTGGTACTT 61.9 ≧60% SEQ ID NO: 43 2 569 588CCAGGCACGTGCACTGGTAC 57.6 ≧55% SEQ ID NO: 176 1 580 599CACCGCCCCGTCCAGGCACG 16.3 SEQ ID NO: 44 1 584 603 AGCCCACCGCCCCGTCCAGG16.2 SEQ ID NO: 45 1 589 608 CATGCAGCCCACCGCCCCGT 23.9 SEQ ID NO: 46 1600 619 CTGCACAGGGGCATGCAGCC 19.7 SEQ ID NO: 47 1 608 627CGTCCATGCTGCACAGGGGC 0.2 SEQ ID NO: 48 1 613 632 ACGAACGTCCATGCTGCACA56.6 SEQ ID NO: 49 1 629 648 AGTCAGGGCTGGGCAGACGA 31.6 SEQ ID NO: 50 1639 658 GGGAAGGGGCAGTCAGGGCT 13.4 SEQ ID NO: 51 1 646 665CCTCCTCGGGAAGGGGCAGT 3.2 SEQ ID NO: 52 1 657 676 GGCAGCTTGACCCTCCTCGG29.0 SEQ ID NO: 53 1 661 680 CCCGGGCAGCTTGACCCTCC 44.7 SEQ ID NO: 54 1677 696 ACTCCTCGCAGCATTTCCCG 29.6 SEQ ID NO: 55 1 684 703CACACCCACTCCTCGCAGCA 51.1 SEQ ID NO: 56 1 688 707 GTCACACACCCACTCCTCGC17.8 SEQ ID NO: 57 1 692 711 GCTCGTCACACACCCACTCC 44.3 SEQ ID NO: 58 1710 729 CCACGGTTTGGTCCTTGGGC 34.5 SEQ ID NO: 59 2 719 738CAGGCCCAACCACGGTTTGG 44.5 SEQ ID NO: 177 1 721 740 GGCAGGCCCAACCACGGTTT

SEQ ID NO: 60 2 723 742 AGGGCAGGCCCAACCACGGT 30.9 SEQ ID NO: 178 1 737756 GTCGGTAAGCCGCGAGGGCA 28.5 SEQ ID NO: 61 2 745 764GTCTTCCAGTCGGTAAGCCG 30.9 SEQ ID NO: 179 1 747 766 GTGTCTTCCAGTCGGTAAGC

SEQ ID NO: 62 2 749 768 ACGTGTCTTCCAGTCGGTAA 1.2 SEQ ID NO: 180 1 754773 GCCAAACGTGTCTTCCAGTC 31.4 SEQ ID NO: 63 1 762 781GGGTCTGGGCCAAACGTGTC 12.5 SEQ ID NO: 64 1 772 791 AATCATAGTIGGGTCTGGGC33.2 SEQ ID NO: 65 2 787 806 CAGGCAGTTGGCTCTAATCA 48.0 SEQ ID NO: 181 2789 808 ACCAGGCAGTTGGCTCTAAT 56.5 ≧55% SEQ ID NO: 182 1 791 810GGACCAGGCAGTTGGCTCTA

≧65% SEQ ID NO: 66 2 793 812 CTGGACCAGGCAGTTGGCTC

≧65% SEQ ID NO: 183 2 795 814 GTCTGGACCAGGCAGTTGGC 55.4 ≧55%SEQ ID NO: 184 3 797 816 TGGTCTGGACCAGGCAGTTG 32.3 SEQ ID NO: 252 1 809828 CGCTCCACTCTGTGGTCTGG 34.6 SEQ ID NO: 67 1 817 836GGAACAGGCGCTCCACTCTG 47.7 SEQ ID NO: 68 1 836 855 TGCCCATCCCACAGGTCTTG9.1 SEQ ID NO: 69 1 841 860 GGAGATGCCCATCCCACAGG 36.8 SEQ ID NO: 70 2855 874 TTGGTAACCCGGGTGGAGAT 30.0 SEQ ID NO: 185 2 857 876CATTGGTAACCCGGGTGGAG 42.0 SEQ ID NO: 186 1 859 878 GTCATTGGTAACCCGGGTGG58.7 ≧55% SEQ ID NO: 71 2 861 880 TTGTCATTGGTAACCCGGGT 55.4 ≧55%SEQ ID NO: 187 2 863 882 CGTTGTCATTGGTAACCCGG 31.5 SEQ ID NO: 188 1 865884 GGCGTTGTCATTGGTAACCC 44.0 SEQ ID NO: 72 1 870 889CAGGAGGCGTTGTCATTGGT 29.4 SEQ ID NO: 73 1 889 908 GCTCTGCTTCTCTAGCCTGC13.8 SEQ ID NO: 74 1 893 912 GGCGGCTCTGCTTCTCTAGC 51.6 ≧50%SEQ ID NO: 75 1 904 923 GACCATGCACAGGCGGCTCT 50.4 ≧50% SEQ ID NO: 76 2908 927 GCCTGACCATGCACAGGCGG 37.8 SEQ ID NO: 189 2 910 929AGGCCTGACCATGCACAGGC 0.5 SEQ ID NO: 190 1 912 931 CAAGGCCTGACCATGCACAG55.3 SEQ ID NO: 77 2 914 933 CGCAAGGCCTGACCATGCAC 36.2 SEQ ID NO: 191 2916 935 TTCGCAAGGCCTGACCATGC 60.0 SEQ ID NO: 192 1 931 950CTCTTCCAGGTCAGCTTCGC 27.1 SEQ ID NO: 78 1 935 954 TGTTCTCTTCCAGGTCAGCT51.6 ≧50% SEQ ID NO: 79 1 946 965 GCCCTTCTTAATGTTCTCTT 62.7 ≧60%SEQ ID NO: 80 3 948 967 TTGCCCTTCTTAATGTTCTC

≧70% SEQ ID NO: 253 3 950 969 TTTTGCCCTTCTTAATGTTC 55.3 ≧55%SEQ ID NO: 254 3 952 971 CTTTTTGCCCTTCTTAATGT 53.1 ≧50% SEQ ID NO: 255 3954 973 CACTTTTTGCCCTTCTTAAT 59.4 ≧55% SEQ ID NO: 256 2 956 975TGCACTTTTTGCCCTTCTTA

≧75% SEQ ID NO: 193 2 958 977 GATGCACTTTTTGCCCTTCT

≧80% SEQ ID NO: 194 1 960 979 CGGATGCACTTTTTGCCCTT

≧65% SEQ ID NO: 81 2 962 981 TACGGATGCACTTTTTGCCC

≧75% SEQ ID NO: 195 2 965 984 GAGTACGGATGCACTTTTTG

≧75% SEQ ID NO: 196 1 967 986 GGGAGTACGGATGCACTTTT

≧70% SEQ ID NO: 82 2 969 988 TTGGGAGTACGGATGCACTT

≧65% SEQ ID NO: 197 2 971 990 TTTTGGGAGTACGGATGCAC 47.8 SEQ ID NO: 198 3976 995 GGAGATTTTGGGAGTACGGA 31.4 SEQ ID NO: 257 3 979 998CTTGGAGATTTTGGGAGTAC 41.1 SEQ ID NO: 258 1 981 1000 GGCTTGGAGATTTTGGGAGT58.2 ≧55% SEQ ID NO: 83 3 983 1002 TAGGCTTGGAGATTTTGGGA 60.3 ≧60%SEQ ID NO: 259 2 987 1006 TTGATAGGCTTGGAGATTTT 49.2 SEQ ID NO: 199 1 9891008 ACTTGATAGGCTTGGAGATT 64.1 ≧60% SEQ ID NO: 84 2 991 1010AAACTTGATAGGCTTGGAGA

≧65% SEQ ID NO: 200 3 993 1012 TCAAACTTGATAGGCTTGGA 39.6 SEQ ID NO: 2601 995 1014 GCTCAAACTTGATAGGCTTG 38.7 SEQ ID NO: 85 1 1003 1022GCCAGAAAGCTCAAACTTGA 38.1 SEQ ID NO: 86 2 1004 1023 AGCCAGAAAGCTCAAACTTG21.9 SEQ ID NO: 201 1 1006 1025 GCAGCCAGAAAGCTCAAAT 62.4 ≧60%SEQ ID NO: 87 2 1008 1027 GTGCAGCCAGAAAGCTCAAA 56.2 ≧55% SEQ ID NO: 2023 1011 1030 CTGGTGCAGCCAGAAAGCTC

≧65% SEQ ID NO: 261 3 1013 1032 TGCTGGTGCAGCCAGAAAGC

≧65% SEQ ID NO: 262 2 1015 1034 CATGCTGGTGCAGCCAGAAA

≧65% SEQ ID NO: 203 2 1017 1036 TTCATGCTGGTGCAGCCAGA

≧80% SEQ ID NO: 204 1 1019 1038 TCTTCATGCTGGTGCAGCCA

≧70% SEQ ID NO: 88 2 1021 1040 TGTCTTCATGCTGGTGCAGC

≧70% SEQ ID NO: 205 2 1023 1042 TATGTCTTCATGCTGGTGCA

≧75% SEQ ID NO: 206 3 1025 1044 GGTATGTCTTCATGCTGGTG 52.3 ≧50%SEQ ID NO: 263 3 1027 1046 TCGGTATGTCTTCATGCTGG 44.1 SEQ ID NO: 264 11029 1048 GCTCGGTATGTCTTCATGCT 47.0 SEQ ID NO: 89 3 1032 1051TTAGCTCGGTATGTCTTCAT 50.0 ≧50% SEQ ID NO: 265 2 1034 1053ATTTAGCTCGGTATGTCTTC

≧65% SEQ ID NO: 207 1 1036 1055 GAATTTAGCTCGGTATGTCT

≧65% SEQ ID NO: 90 2 1039 1058 ACAGAATTTAGCTCGGTATG −11.2 SEQ ID NO: 2083 1041 1060 CCACAGAATTTAGCTCGGTA 29.8 SEQ ID NO: 266 1 1043 1062CTCCACAGAATTTAGCTCGG

SEQ ID NO: 91 2 1045 1064 TACTCCACAGAATTTAGCTC 42.4 SEQ ID NO: 209 21055 1074 CGTCGGTACATACTCCACAG 39.5 SEQ ID NO: 210 1 1057 1076GCCGTCGGTACATACTCCAC

SEQ ID NO: 92 2 1059 1078 CGGCCGTCGGTACATACTCC 35.5 SEQ ID NO: 211 21061 1080 ATCGGCCGTCGGTACATACT 57.6 ≧55% SEQ ID NO: 212 2 1064 1083AGCATCGGCCGTCGGTACAT

≧65% SEQ ID NO: 213 2 1066 1085 GCAGCATCGGCCGTCGGTAC

≧70% SEQ ID NO: 214 1 1068 1087 GTGCAGCATCGGCCGTCGGT

≧65% SEQ ID NO: 93 2 1070 1089 GGGTGCAGCATCGGCCGTCG

≧70% SEQ ID NO: 215 1 1071 1090 GGGGTGCAGCATCGGCCGTC 49.3 SEQ ID NO: 941 1074 1093 TGGGGGGTGCAGCATCGGCC 37.7 SEQ ID NO: 95 1 1081 1100GGTTCTGTGGGGGGTGCAGC 22.3 SEQ ID NO: 96 1 1088 1107 GGGTGGTGGTTCTGTGGGGG−11.4 SEQ ID NO: 97 1 1093 1112 CGGCAGGGTGGTGGTTCTGT 42.0 SEQ ID NO: 982 1105 1124 CTTGAACTCCACCGGCAGGG 46.1 SEQ ID NO: 216 2 1107 1126CACTTGAACTCCACCGGCAG 50.5 ≧50% SEQ ID NO: 217 1 1109 1128GGCACTTGAACTCCACCGGC

≧65% SEQ ID NO: 99 2 1111 1130 AGGGCACTTGAACTCCACCG

≧65% SEQ ID NO: 218 2 1113 1132 TCAGGGCACTTGAACTCCAC

≧65% SEQ ID NO: 219 3 1116 1135 CCGTCAGGGCACTTGAACTC 60.7 ≧60%SEQ ID NO: 267 2 1118 1137 CGCCGTCAGGGCACTTGAAC

≧70% SEQ ID NO: 220 2 1120 1139 CTCGCCGTCAGGGCACTTGA

≧70% SEQ ID NO: 221 1 1122 1141 ACCTCGCCGTCAGGGCACTT

≧65% SEQ ID NO: 100 2 1124 1143 TGACCTCGCCGTCAGGGCAC

≧75% SEQ ID NO: 222 2 1126 1145 CATGACCTCGCCGTCAGGGC

≧65% SEQ ID NO: 223 3 1128 1147 TTCATGACCTCGCCGTCAGG 38.3 SEQ ID NO: 2683 1130 1149 TCTTCATGACCTCGCCGTCA 44.1 SEQ ID NO: 269 1 1132 1151CTTCTTCATGACCTCGCCGT 59.8 ≧55% SEQ ID NO: 101 3 1134 1153TTCTTCTTCATGACCTCGCC 51.0 ≧50% SEQ ID NO: 270 1 1142 1161ACATCATGTTCTTCTTCATG −4.2 SEQ ID NO: 102 2 1157 1176CACAGGTCTTGATGAACATC 44.7 SEQ ID NO: 224 1 1159 1178GGCACAGGTCTTGATGAACA 64.4 SEQ ID NO: 103 2 1161 1180CAGGCACAGGTCTTGATGAA 46.5 SEQ ID NO: 225 3 1164 1183TGGCAGGCACAGGTCTTGAT 52.6 ≧50% SEQ ID NO: 271 2 1166 1185AATGGCAGGCACAGGTCTTG

≧65% SEQ ID NO: 226 1 1168 1187 GTAATGGCAGGCACAGGTCT

≧65% SEQ ID NO: 104 2 1170 1189 TTGTAATGGCAGGCACAGGT 60.9 ≧60%SEQ ID NO: 227 3 1172 1191 AGTTGTAATGGCAGGCACAG 52.8 ≧50% SEQ ID NO: 2722 1175 1194 GACAGTTGTAATGGCAGGCA 62.0 ≧60% SEQ ID NO: 228 2 1177 1196GGGACAGTTGTAATGGCAGG 36.4 SEQ ID NO: 229 1 1179 1198CCGGGACAGTTGTAATGGCA

≧65% SEQ ID NO: 105 1 1181 1200 CTCCGGGACAGTTGTAATGG 64.3 ≧60%SEQ ID NO: 230 2 1183 1202 GTCTCCGGGACAGTTGTAAT

≧65% SEQ ID NO: 231 3 1185 1204 TTGTCTCCGGGACAGTTGTA

≧65% SEQ ID NO: 273 3 1187 1260 CATTGTCTCCGGGACAGTTG

≧70% SEQ ID NO: 274 3 1189 1208 GTCATTGTCTCCGGGACAGT 61.3 ≧60%SEQ ID NO: 275 2 1191 1210 ATGTCATTGTCTCCGGGACA

≧70% SEQ ID NO: 232 1 1193 1212 AGATGTCATTGTCTCCGGGA

≧65% SEQ ID NO: 106 2 1195 1214 AAAGATGTCATTGTCTCCGG

≧65% SEQ ID NO: 233 3 1197 1216 TCAAAGATGTCATTGTCTCC 51.8 ≧50%SEQ ID NO: 276 3 1199 1218 ATTCAAAGATGTCATTGTCT 42.4 SEQ ID NO: 277 31201 1220 CGATTCAAAGATGTCATTGT 12.2 SEQ ID NO: 278 3 1203 1222AGCGATTCAAAGATGTCATT 55.7 SEQ ID NO: 279 1 1205 1224ACAGCGATTCAAAGATGTCA 31.9 SEQ ID NO: 107 3 1209 1228TAGTACAGCGATTCAAAGAT 38.7 SEQ ID NO: 280 1 1211 1230TGTAGTACAGCGATTCAAAG 58.5 ≧55% SEQ ID NO: 108 3 1213 1232CCTGTAGTACAGCGATTCAA 61.2 ≧60% SEQ ID NO: 281 1 1228 1247GTCTCCGTACATCTTCCTGT 55.7 ≧55% SEQ ID NO: 109 1 1233 1252GCCATGTCTCCGTACATCTT 40.0 SEQ ID NO: 110 3 1236 1255CATGCCATGTCTCCGTACAT 55.6 ≧55% SEQ ID NO: 282 2 1238 1257tTCATGCCATGTCTCCGTAC

≧70% SEQ ID NO: 234 1 1240 1259 gctTCATGCCATGTCTCCGT

≧65% SEQ ID NO: 111 2 1242 1261 tggctTCATGCCATGTCTCC

≧70% SEQ ID NO: 235 3 1244 1263 tctggctTCATGCCATGTCT 53.0 ≧50%SEQ ID NO: 283 3 1246 1265 tctctggctTCATGCCATGT

≧65% SEQ ID NO: 284 1 1249 1268 cactctctggctTCATGCCA 36.3 SEQ ID NO: 1121 1255 1274 gtctctcactctctggctTC 46.7 SEQ ID NO: 113 1 1261 1280gttaatgtctctcactctct 42.5 SEQ ID NO: 114 1 1286 1305atcagttcaagttccagtct 43.8 SEQ ID NO: 115 1 1305 1324acggaaaaatgagatgtgaa 17.3 SEQ ID NO: 116 1 1320 1339actgaaatcatttttacgga −0.7 SEQ ID NO: 117 3 1327 1346ttgtgctactgaaatcattt 37.6 SEQ ID NO: 285 1 1329 1348acttgtgctactgaaatcat 58.3 ≧55% SEQ ID NO: 118 3 1331 1350taacttgtgctactgaaatc 59.6 ≧55% SEQ ID NO: 286 1 1357 1376cccccagttagaaaaacaga 35.5 SEQ ID NO: 119 1 1366 1385gaatcttttcccccagttag 47.1 SEQ ID NO: 120 1 1384 1403atgttttgaattgggtggga 5.5 SEQ ID NO: 121 1 1405 1424 ctatttgtttgacatggcac43.0 SEQ ID NO: 122 1 1417 1436 ggggttgatagactatttgt 9.9 SEQ ID NO: 1231 1425 1444 cagtgtctggggttgataga 43.3 SEQ ID NO: 124 1 1437 1456cattcttcaaaccagtgtct 14.0 SEQ ID NO: 125 1 1455 1474tccactgtcaagtcttaaca 23.7 SEQ ID NO: 126 1 1460 1479gtagttccactgtcaagtct 49.7 SEQ ID NO: 127 1 1483 1502acattctggtgctgtgtact 48.4 SEQ ID NO: 128 1 1498 1517gccacaccttaatatacatt 48.2 SEQ ID NO: 129 1 1514 1533tcccactgctcctaaagcca 56.3 ≧55% SEQ ID NO: 130 3 1516 1535cctcccactgctcctaaagc 59.5 ≧55% SEQ ID NO: 287 2 1518 1537accctcccactgctcctaaa

≧65% SEQ ID NO: 236 1 1520 1539 gtaccctcccactgctccta 64.5 ≧60%SEQ ID NO: 131 3 1522 1541 tggtaccctcccactgctcc 57.4 ≧55% SEQ ID NO: 2882 1524 1543 gctggtaccctcccactgct

≧65% SEQ ID NO: 237 1 1527 1546 tctgctggtaccctcccact 64.4 ≧60%SEQ ID NO: 132 2 1529 1548 tttctgctggtaccctccca 53.8 ≧50% SEQ ID NO: 2381 1531 1550 cctttctgctggtaccctcc 59.4 ≧55% SEQ ID NO: 133 3 1533 1552aacctttctgctggtaccct

≧65% SEQ ID NO: 289 1 1549 1568 gctatctgatgatactaacc 46.0 SEQ ID NO: 1341 1574 1593 agcaggcatattactcgtat 52.0 SEQ ID NO: 135 1 1585 1604acacttcaaatagcaggcat 41.0 SEQ ID NO: 136 1 1597 1616tccttctcaattacacttca 14.9 SEQ ID NO: 137 3 1617 1636cagtgagcacgctaaaattt 54.0 SEQ ID NO: 290 1 1620 1639ggtcagtgagcacgctaaaa 40.3 SEQ ID NO: 138 3 1623 1642gcaggtcagtgagcacgcta 62.6 ≧60% SEQ ID NO: 291 3 1625 1644aggcaggtcagtgagcacgc 64.8 ≧60% SEQ ID NO: 292 3 1627 1646acaggcaggtcagtgagcac 56.2 ≧55% SEQ ID NO: 293 2 1629 1648ctacaggcaggtcagtgagc 62.5 ≧60% SEQ ID NO: 239 2 1631 1650ggctacaggcaggtcagtga

≧65% SEQ ID NO: 240 1 1633 1652 ggggctacaggcaggtcagt

≧65% SEQ ID NO: 139 2 1635 1654 ctggggctacaggcaggtca 62.5 ≧60%SEQ ID NO: 241 2 1637 1656 cactggggctacaggcaggt

≧65% SEQ ID NO: 242 3 1639 1658 gtcactggggctacaggcag 46.9 SEQ ID NO: 2943 1641 1660 ctgtcactggggctacaggc 44.1 SEQ ID NO: 295 3 1643 1662agctgtdactggggctacag 58.2 SEQ ID NO: 296 3 1645 1664ctagctgtcactggggctac 36.3 SEQ ID NO: 297 1 1647 1666tcctagctgtcactggggct 48.3 SEQ ID NO: 140 3 1649 1668catcctagctgtcactgggg 49.6 SEQ ID NO: 298 2 1651 1670cacatcctagctgtcactgg 56.0 ≧55% SEQ ID NO: 243 1 1653 1672tgcacatcctagctgtcact 60.4 ≧60% SEQ ID NO: 141 2 1655 1674aatgcacatcctagctgtca 43.5 SEQ ID NO: 244 1 1670 1689tcttgatggctggagaatgc 49.3 SEQ ID NO: 142 1 1681 1700ttgactcagtctcttgatgg 10.1 SEQ ID NO: 143 1 1712 1731ctgagtctgctgttctgact 30.9 SEQ ID NO: 144 1 1740 1759cagtgtcattcgaatcagaa 37.4 SEQ ID NO: 145 3 1750 1769gattcctgaacagtgtcatt 53.9 ≧50% SEQ ID NO: 299 1 1752 1771ccgattcctgaacagtgtca 59.5 ≧55% SEQ ID NO: 146 3 1755 1774attccgattcctgaacagtg 50.2 ≧50% SEQ ID NO: 300 3 1757 1776ggattccgattcctgaacag 45.7 SEQ ID NO: 301 3 1759 1778caggattccgattcctgaac 51.1 ≧50% SEQ ID NO: 302 2 1761 1780gacaggattccgattcctga 60.3 ≧60% SEQ ID NO: 245 1 1763 1782tcgacaggattccgattcct 62.8 ≧60% SEQ ID NO: 147 2 1765 1784aatcgacaggattccgattc 65.0 ≧65% SEQ ID NO: 246 3 1767 1786ctaatcgacaggattccgat 55.2 ≧55% SEQ ID NO: 303 3 1769 1788gtctaatcgacaggattccg 45.1 SEQ ID NO: 304 3 1771 1790cagtctaatcgacaggattc 64.5 ≧60% SEQ ID NO: 305 3 1773 1792tccagtctaattgacaggat 59.8 ≧55% SEQ ID NO: 306 3 1775 1794tgtccagtctaatcgacagg

≧65% SEQ ID NO: 307 3 1777 1796 gctgtccagtctaatcgaca 51.0 ≧50%SEQ ID NO: 308 2 1779 1798 aagctgtccagtctaatcga 64.0 ≧60% SEQ ID NO: 2472 1781 1800 acaagctgtccagtctaatc 59.8 ≧55% SEQ ID NO: 248 1 1783 1801ccacaagctgtccagtctaa

≧70% SEQ ID NO: 148 1 1785 1804 tgccacaagctgtccagtct

≧65% SEQ ID NO: 149 2 1787 1806 cttgccacaagctgtccagt 62.9 ≧60%SEQ ID NO: 249 1 1789 1808 cacttgccacaagctgtcca

≧70% SEQ ID NO: 150 2 1791 1810 ttcacttgccacaagctgtc 19.9 SEQ ID NO: 2502 1793 1812 aattcacttgccacaagctg 62.6 ≧60% SEQ ID NO: 251 3 1795 1814caaattcacttgccacaagc 58.7 ≧55% SEQ ID NO: 309 3 1797 1816ggcaaattcacttgccacaa 50.1 ≧50% SEQ ID NO: 310 3 1799 1818caggcaaattcacttgccac 52.2 ≧50% SEQ ID NO: 311 3 1801 1820tacaggcaaattcacttgcc 51.2 ≧50% SEQ ID NO: 312 1 1803 1822gttacaggcaaattcacttg 20.3 SEQ ID NO: 151 3 1891 1910ttaacttagataactgtaca 25.1 SEQ ID NO: 313 1 1892 1911attaacttagataactgtac −3.3 SEQ ID NO: 152 1 1909 1928ggcacaaacaactttaaatt 40.3 SEQ ID NO: 153 1 1962 1981cagaaattgaggctaacatt 16.6 SEQ ID NO: 154 1 1980 1999cattctacctatggtgttca 41.8 SEQ ID NO: 155 1 1986 2005gctttacattctacctatgg 23.7 SEQ ID NO: 156 1 1998 2017acgatcagacaagctttaca 34.5 SEQ ID NO: 157 1 2018 2037gtatccatttcatgctttga 38.1 SEQ ID NO: 158 1 2026 2045ccatataagtatccatttca 22.0 SEQ ID NO: 159 1 2052 1071actgtcattctatctgagca 33.3 SEQ ID NO: 160 1 2056 2075acggactgtcattctatctg 18.8 SEQ ID NO: 161 1 2086 2105atgcctcccctttgcaaaca 21.4 SEQ ID NO: 162 1 2091 2110cactgatgcctcccctttgc 53.8 SEQ ID NO: 163 1 2115 2134acctagaaatcagcctgcca 36.0 SEQ ID NO: 164 1 2131 2150ggctaccacatttcctacct 32.6 SEQ ID NO: 165 1 2153 2172gccatttgttcattaaaagt 11.1 SEQ ID NO: 166 1 2175 2194agtcactcagtttttaataa 19.4 SEQ ID NO: 167 1 2184 2203gctatatagagtcactcagt 21.1 SEQ ID NO: 168 1 2208 2227gcttccaggtgaaaaaactg 22.8 SEQ ID NO: 169 3 2213 2232caaatgcttccaggtgaaaa 37.3 SEQ ID NO: 314 1 2215 2234aacaaatgcttccaggtgaa 55.8 SEQ ID NO: 170 3 2217 2236gaaacaaatgcttccaggtg −0.2 SEQ ID NO: 315 1 2221 2240agtagaaacaaatgcttcca 5.6 SEQ ID NO: 171 1 2243 2262 gtccgaaaaacagtcatatc43.4 SEQ ID NO: 172 1 2258 2277 ctcaacaaataaactgtccg 39.2 SEQ ID NO: 1733 2267 2286 ggtcacactctcaacaaata 46.1 SEQ ID NO: 316 3 2282 2301aaacatgtaacttttggtca 20.7 SEQ ID NO: 317 1 2311 2330atacactttattttcaacta 31.0 SEQ ID NO: 174 Table 2. Combined median valuesfrom 3 or more independent experiments in which the indicated antisenseoligonucleotides were tested. 20-nucleotide-longoligodeoxyribonucleotides with phosphorothioate internucleoside linkagesthroughout the oligonucleotide were assessed for inhibition of CTGF mRNAexpression, expressed here as % inhibition vs. vehicle-treated control.The 5′and 3′ target sites denote the 5′-most and 3′-most complementary(sense) bases of the splice human connective tissue growth factor mRNAsequence (GenBank accession number NM_001901, SEQ ID NO: 1). An “m”suffix denotes an oligonucleotide that crosses an exon-exon boundary andthus is only complementary to the splice mRNA. All other ASOs arecomplementary to both the spliced mRNA and non-spliced pre-mRNA. Thevalues provided in Table 2 also take into account data obtained fromRound 3 experiments for 29 “bridging” ASOs included in Round 1 and Round3 studies as well as “bridging” ASOs included in both Round 2 and Round3 studies. The percent inhibition values for these bridging ASOs werehighly correlated across Rounds (r ≧ 0.81, p < 1e−6).

Example 9 Round 3 Experiments

Based on the results achieved in the Round 2 studies, 66 additional“Round 3” antisense oligonucleotides were chosen to further refine theboundaries of human CTGF mRNA hypersensitive regions from Round 1 andRound 2, or to evaluate other regions not probed as intensely in Round 1or Round 2. Specifically, in choosing new ASOs to test, antisenseoligonucleotides that differed in position by a single base from a Round1 or Round 2 oligonucleotide were avoided in favor of those thatdiffered by at least two of three bases. Additionally, in refininghypersensitive regions, if the potent ASO that defined the end of ahypersensitive region provided >60% CTGF mRNA knockdown in Round 1 orRound 2, then sufficient ASOs were chosen to probe the sequence betweenthe end of the hypersensitive region and the next sequence that wasprobed in Round 1 or Round 2. If the potent ASO that defined the end ofa hypersensitive region provided 55%-60% CTGF mRNA knockdown in Round 1or Round 2, then, a single flanking ASO was tested. If the potent ASOthat defined the end of a hypersensitive region provided <55% KD Round 1or Round 2, then the limits of the hypersensitive region was judgeddefined, and no further testing was performed. Additionally, isolatedpotent ASOs that provided 55-60% KD in Round 1 or Round 2 were tested oneach side with a flanking oligonucleotide that differed by 1 or 2nucleotides.

Each of the Round 3 ASOs were tested in three Round 3 experiments, theresults of which further extended and refined the hypersensitive regionsof the human CTGF mRNA previously identified by ASO knockdown in Rounds1 and 2 (Table 2). Round 3 experiments included 29 Round 1 and 25 Round2 ASOs that served as Round-to-Round “bridging” controls. Thirty threeof the bridging controls conferred >70% CTGF mRNA knockdown (KD) inprior Round 1 or Round 2 experiments. The remaining controls weredefined as “non-specific”, i.e., conferred <34% CTGF mRNA KD. Thesebridging controls produced similar results in the Round 3 experimentscompared to what was observed in prior Rounds (r=0.86, p<1e-16), furtherdemonstrating the reproducibility of the assay system.

As was also seen in Round 2 studies, there was a shift in thedistribution of inhibitory activity for Round 3 ASOs vs Round 1 ASOs,FIGS. 1 and 5, with a significantly higher proportion of Round 3 ASOs(65%) eliciting ≧50% CTGF mRNA knockdown than Round 1 ASOs (25%,p=1e-8). These data further demonstrate that potent ASOs are notrandomly distributed along human CTGF mRNA, but instead are clustered indefined regions that are hypersensitive to ASO-mediated knockdown. Thisclustering of hypersensitive regions can be seen in FIGS. 6A-6E. Thesequences and SEQ ID NOs of the identified hypersensitive regions ofhuman CTGF mRNA are listed in Table 3.

Example 10 Hypersensitive Regions of CTGF mRNA

In total 316 unique 20-mer ASOs were tested to identify ASOs with highinhibitory activity and to define the discovered hypersensitive regionsof human CTGF mRNA. These hypersensitive regions can be discerned frominspection of the Regional Status column of Table 2. In Table 3, thesequence of each hypersensitive region is further provided along withits respective SEQ ID NO and the overall percent inhibition of CTGF mRNAexpression associated with the specific region.

TABLE 3 Hypersensitive Regions of human CTGF mRNA Nucleotide RangeHypersensitive Sequence SEQ ID NO. % Inhibition 567 to 588aagtaccagt gcacgtgcct gg SEQ ID NO: 318 ≧55% 789 to 414attagagcca actgcctggt ccagac SEQ ID NO: 319 ≧55% 859 to 880ccacccgggt taccaatgac aa SEQ ID NO: 320 ≧55% 946 to 988aagagaacat taagaagggc SEQ ID NO: 321 ≧50% aaaaagtgca tccgtactcc caa 981 to 1002 actcccaaaa tctccaagcc ta SEQ ID NO: 322 ≧55%  989 to 1010aatctccaag cctatcaagt tt SEQ ID NO: 323 ≧60% 1006 to 1044agtttgagct ttctggctgc SEQ ID NO: 324 ≧50% accagcatga agacatacc1032 to 1055 atgaagacat accgagctaa attc SEQ ID NO: 325 ≧50% 1061 to 1089agtatgtacc gacggccgat gctgcaccc SEQ ID NO: 326 ≧55% 1107 to 1145ctgccggtgg agttcaagtg SEQ ID NO: 327 ≧50% ccctgacggc gaggtcatg1132 to 1153 acggcgaggt SEQ ID NO: 328 ≧50% catgaagaag aa 1164 to 1194atcaagacct gtgcctgcca SEQ ID NO: 329 ≧50% ttacaactgt c 1179 to 1216tgccattaca actgtcccgg SEQ ID NO: 330 ≧50% agacaatgac atctttga1211 to 1232 ctttgaatcg ctgtactaca gg SEQ ID NO: 331 ≧55% 1236 to 1265atgtacggag acatggcatg SEQ ID NO: 332 ≧50% aagccagaga 1329 to 1350atgatttcag tagcacaagt ta SEQ ID NO: 333 ≧55% 1514 to 1552tggctttagg agcagtggga SEQ ID NO: 334 ≧50% gggtaccagc agaaaggtt1623 to 1656 tagcgtgctc actgacctgc SEQ ID NO: 335 ≧55% ctgtagcccc agtg1651 to 1672 ccagtgacag ctaggatgtg ca SEQ ID NO: 336 ≧55% 1750 to 1774aatgacactg ttcaggaatc ggaat SEQ ID NO: 337 ≧50% 1759 to 1786gttcaggaat cggaatcctg tcgattag SEQ ID NO: 338 ≧50% 1771 to 1808gaatcctgtc gattagactg SEQ ID NO: 339 ≧50% gacagcttgt ggcaagtg1793 to 1820 cagcttgtgg caagtgaatt tgcctgta SEQ ID NO: 340 ≧50% Table 3.Nucleotide ranges correspond to published human CTGF mRNA sequence(GenBank accession number NM_001901, SEQ ID NO: 1), wherein thymidine issubstituted for uridine in the sequence. Sequences and nucleotide rangesare provided in the sense orientation.

The percent inhibition listed for each hypersensitivity region in Table3 is the lowest value found in a particular hypersensitivity region.Most of the hypersensitive regions contain sub-regions that demonstratedhigher sensitivity to antisense mediated inhibition. For example, thehypersensitive region from nucleotides 1006 to 1044, SEQ ID NO: 324,demonstrated at least 50% inhibition, but it has a sub-region fromnucleotides 1017 to 1042 that demonstrated at least 70% inhibition.

Example 11 Additional Human Cell Lines Tested

The ability to broadly apply these findings across cell types andbiological systems was demonstrated by retesting a subset of potent andnon-potent antisense oligonucleotides in human MG63 osteosarcoma andA549 lung adenocarcinoma cell lines. Two independent transfectionexperiments were performed per cell line. The results demonstratedexcellent correlations between the level of reduction of CTGF mRNAexpression provided by the respective ASOs in Hs578T versus A549(r=0.93), Hs578T versus MG63 (r=0.88), and MG63 versus A549 cells(r=0.81) (p>1e-7 each). These correlation studies showed that mRNAinhibition in Hs578T cells could be used to predict inhibitory activityin disparate cell types, indicating that hypersensitive regions arepreserved across different CTGF expressing, cell types. These resultsdemonstrate that different cell types share the identifiedhypersensitive regions of human CTGF mRNA and support the use of theantisense oligonucleotides of the invention for the treatment of allCTGF associated disorders and conditions.

Example 12 Similar Results in a Murine Cell Line

The CTGF mRNA hypersensitive regions identified by ASOs in human celllines appear to be similar in CTGF mRNA from other mammals as well. Thisis demonstrated by the observation that ASOs that have identical humanand murine target sequences (perfect complementarity) caused a median %KD in murine C2C12 cells that was significantly correlated median % KDseen in human Hs578T cells (r=0.78, p<4e-10).

Example 13 Inhibition of CTGF Protein Production

The ability of potent ASOs to alter the secretion of human CTGF proteinis tested in Hs578T cells. As outlined in Example 3, cells are seededinto 96-well plates at 2000 cells/well in 100 μL of Hs578T growthmedium. The cells are grown for 24 hrs in a humidified 37° C. incubatorwith 5% CO₂ after which the growth medium is removed and replaced with80 μL of fresh growth medium. Designated wells then receive 20 μL ofserum-free Opti-MEM® containing a potent or non-potent ASO and 0.5 μL ofDharmaFECT® 4 transfection reagent as per the manufacturer'sinstructions. The final ASO concentration is 150 mM and each ASO istested in three independent wells.

Cells are cultured for 17 hrs, after which 1 μL of DMEM containing 5mg/mL heparin (Sigma-Aldrich Co., St. Louis, Mo.) is added to each well.The cells are then cultured for 1 hr, after which the growth medium isremoved and the cells are washed once with 100 μL of PBS. After the PBSis removed, 100 μL of growth medium with 50 μg/mL heparin is added toeach well. Cells are then cultured for 8 hrs, after which the culturemedium is collected and frozen at −80° C. The cells are then re-fed with100 μL of fresh growth medium containing 50 μg/mL heparin and culturedfor an additional 16 hrs, after which the new growth medium is collectedand stored at −80° C.

The amount of CTGF protein secreted by the cells during the intervalsfrom 0 to 8 hrs and 8 to 24 hrs after the end of the 18 hr transfectionperiod is measured by sandwich ELISA using monoclonal antibodies againstepitopes of C- and N-terminal CTGF as capture and secondary antibodyusing recombinant CTGF as the standard (Dziadzio et al. Q J Med 98:485-,2005). Treatment with the three potent ASOs result in lower levels ofCTGF secretion at each time interval compared to the treatment with thethree non-potent ASOs.

Example 14 Murine Cutaneous Wound-Healing Model

Mice are anesthetized with rodent cocktail (0.075 mg ketamine/0.015 mgxylazine/0.0025 mg aceprozamine per gram weight of mouse). The backs areshaved and disinfected. A full-thickness skin excision is made on thedorsal midline using an 8-mm dermal biopsy punch. The wounds are leftopen, but dressed.

Antisense Oligonucleotide Treatment

Mice are randomly divided into two groups (n=10 for each). On Day 0, onegroup is treated with 100 μg of antisense oligonucleotide, SEQ ID NO:204, formulated in a slow release dressing. The other group receives aslow release dressing devoid of the antisense oligonucleotide as acontrol. Mice are monitored daily for general health and to check theintegrity of the dressings. Mice are weighed twice weekly starting onDay −1.

On Day 15 post-biopsy, the animals are sacrificed. A sample of skin fromthe center of each wound is obtained with a 0.5 cm biopsy punch, andmRNA is extracted using standard procedures. RT-PCR mRNA analysis ofmouse CTGF and Colla2 is performed with mouse β-actin (Actb) used as thenormalization gene.

Results

Treatment results in a statistically significant reduction in both CTGFand Colla2 mRNA expression and demonstrates that inhibition of CTGF mRNAexpression with an antisense oligonucleotide will decrease thedeposition of collagen in skin, and hence, reduce the severity of scarformation. The change in weight of the treated mice group is notstatistically different from the untreated group indicating thenon-toxic nature of the treatment.

Example 15 Murine Surgical Adhesion Model

A hyaluronan hydrogel (Yeo et al. Ann Surg. 2007 May; 245(5):819-24) isused in the murine surgical model (Gorvy et al. Am J Pathol. 2005October; 167(4):1005-19) to demonstrate the ability of an antisenseoligonucleotide to inhibit the formation of surgical adhesions.

Animals

C57BL/6J adult male mice, age 10 to 12 weeks with a weight of 25 to 30g, are used. Mice are maintained under standard conditions of food andwater ad libitum on a 12-hour day-night cycle. Prior to surgery, miceare randomly divided into two groups, sham treated and treated (n=6 foreach). Mice are then anesthetized with a mixture of inhaled isofluraneand oxygen, and a midline incision is made through the abdominal walland peritoneum. A standard site (6 mm diameter and 1 mm depth), midwayand ˜0.5 cm lateral to the midline incision on the left abdominal wall,is injured using a trauma instrument (Dr. Mark Eastwood, Department ofBiomedical Sciences, University of Westminster, London, UK). The cecumis isolated and scraped 30 times on its lateral aspect with a scalpelblade, after first irrigating with 0.9% sterile saline. Hemorrhage isinduced by lacerating a small blood vessel on the medial surface of thececum with a hypodermic needle. The two injured surfaces are thenapposed by placing two horizontal mattress sutures (8/0 Ethilon;Ethicon, Berkshire, UK) 1.2 cm apart.

Antisense Oligonucleotide Treatment

Sham treated mice receive 2 ml of cross-linked hydrogel placed betweenthe two injured surfaces, while antisense oligonucleotide treated micereceive 2 ml of cross-linked hydrogel containing 100 μg (50 μg/ml) ofthe antisense oligonucleotide represented by SEQ ID NO: 204. The midlineincision is closed in two layers; the linea-alba with a continuoussuture, and then the skin using interrupted sutures (6/0 Ethilon,Ethicon, Somerville, N.J.).

At 7 days after surgery, mice are sacrificed and adhesions are assessedmacroscopically according to the number of adhesions and sites ofadhesion formation. Additionally, the mice are photographed forindependent examination. A category 1 adhesion is defined as an adhesionbetween the two abraded serosal surfaces, i.e., cecum to abraded bodywall. Category 2 adhesions involve the two abraded surfaces and anuninvolved site, such as the greater omentum and abraded body wall.Category 3 adhesions describe adhesions that form at a distant site tothe abraded serosa, for example the fat body to the midline incision.

Results

All animals in the sham treated group form adhesions at the trauma site(category 1) and with some adhesions noted between abraded surfaces andan uninvolved site (category 2) and at distant sites (category 3) areobserved in 2 animals. In contrast, animals treated with the antisenseoligonucleotide containing hydrogel show significant in trauma siteadhesions and adhesions between abraded surfaces and uninvolved siteswith no distant site adhesions present.

What is claimed:
 1. A modified antisense oligonucleotide, 12 to 20nucleotides in length, that is complementary to a region of humanconnective tissue growth factor (CTGF) mRNA and comprises at least 12consecutive nucleotides of an oligonucleotide selected from the groupconsisting of SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO:232, SEQ ID NO: 106, SEQ ID NO: 233 and SEQ ID NO:
 276. 2. A modifiedantisense oligonucleotide between 12 and 30 nucleotides in length thatis complementary to a region within nucleotides 1185 to 1216 of SEQ IDNO:1.
 3. The oligonucleotide of claim 1 or 2, wherein the modificationof the oligonucleotide comprises the incorporation of at least onenon-naturally occurring internucleoside linkage, sugar moiety, ornucleobase.
 4. The oligonucleotide of claim 3, wherein the at least onenon-naturally occurring internucleoside linkage is a phosphothioateinternucleoside linkage.
 5. The oligonucleotide of claim 4, wherein allof the internucleoside linkages are phosphothioate internucleosidelinkages.
 6. The oligonucleotide of claim 3, wherein the at least onenon-naturally occurring sugar moiety is a bicyclic sugar.
 7. Theoligonucleotide of claim 3, wherein the at least one non-naturallyoccurring sugar comprises a 2′-O,4′-C-methylene bridge.
 8. Theoligonucleotide of claim 3, wherein the at least one non-naturallyoccurring nucleobase is a 5-methylcytosine.
 9. The modified antisenseoligonucleotide of claim 1 that is complementary to a region of humanconnective tissue growth factor (CTGF) mRNA and comprises SEQ ID NO:274.10. A modified antisense oligonucleotide selected from the groupconsisting of SEQ ID NO: 105, SEQ ID NO: 230, SEQ ID NO 231, SEQ ID NO:273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 232, SEQ ID NO: 106, SEQID NO: 233 and SEQ ID NO:
 276. 11. The modified antisenseoligonucleotide of claim 2, wherein the modified antisenseoligonucleotide is between 12 and 20 nucleotides in length.
 12. A methodof treating a CTGF-associated disorder comprising, administering to asubject in need thereof, an effective amount of a modified antisenseoligonucleotide, 12 to 30 nucleotides in length, that is complementaryto a region within nucleotides 1185 to 1216 of SEQ ID NO: 1, therebytreating the CTGF-associated disorder.
 13. The method of claim 12,wherein the CTGF-associated disorder is selected from the groupconsisting of dermal fibrosis, liver fibrosis, pulmonary fibrosis, renalfibrosis, cardiac fibrosis, ocular fibrosis, scleroderma, surgical scarsand adhesions, scars from wounds, scars from burns, restenosis,glomerular sclerosis, osteoarthritis and cancer.
 14. The method of claim13, wherein the cancer is selected from the group consisting of acutelymphoblastic leukemia, dermatofibromas, breast cancer, breast carcinomadesmoplasia, angiolipoma, angioleiomyoma, desmoplastic cancer, prostatecancer, ovarian cancer, colorectal cancer, pancreatic cancer,gastrointestinal cancer, and liver cancer.